Gene expression in Lucilia sericata (Diptera: Calliphoridae) larvae exposed to Pseudomonas aeruginosa and Acinetobacter baumannii identifies shared and microbe-specific induction of immune genes.

Antibiotic resistance is a continuing challenge in medicine. There are various strategies for expanding antibiotic therapeutic repertoires, including the use of blow flies. Their larvae exhibit strong antibiotic and antibiofilm properties that alter microbiome communities. One species, Lucilia sericata, is used to treat problematic wounds due to its debridement capabilities and its excretions and secretions that kill some pathogenic bacteria. There is much to be learned about how L. sericata interacts with microbiomes at the molecular level. To address this deficiency, gene expression was assessed after feeding exposure (1 h or 4 h) to two clinically problematic pathogens: Pseudomonas aeruginosa and Acinetobacter baumannii. The results identified immunity-related genes that were differentially expressed when exposed to these pathogens, as well as non-immune genes possibly involved in gut responses to bacterial infection. There was a greater response to P. aeruginosa that increased over time, while few genes responded to A. baumannii exposure, and expression was not time-dependent. The response to feeding on pathogens indicates a few common responses and features distinct to each pathogen, which is useful in improving the wound debridement therapy and helps to develop biomimetic alternatives.

Acari community in association with delayed pig carrion decomposition.

Acari community structure and function associated with delayed pig carrion decomposition has not been examined. In this study, 18 swine carcasses were studied in central Texas, USA, during two consecutive summers (2013 and 2014). Samples of ca. 400 g soil were collected from beneath, aside, and 5 m away from each pig carcass over 180 days. Mites from soil samples were extracted using Berlese funnels and identified to order and family levels and classified according to ecological function. In total 1565 and 1740 mites were identified from the 2013 and 2014 soil samples, respectively. Significant differences were determined for mite community structure at order and family levels temporally on carrion (e.g., day 0 × day 14) regardless of treatments and between soil regions where mites were collected (e.g., soil beneath vs. soil 5 m away from carrion). However, no significant differences were found in mite community structure at the order level between pig carrion with and without delayed Diptera colonization (i.e., treatments). Analysis at the family level determined a significant difference across treatments for both summers. Ecological function of mites did not change significantly following the delayed decomposition of pig carcasses. However, detritivores and fungivores were significant indicator groups during the pig carrion decomposition process. Furthermore, 13 phoretic mite species associated with eight forensically important beetle species were documented. Data from this study indicated that the rate of nutrient flow into the soil impacted associated arthropod communities; however, detecting such shifts depends on the taxonomic resolution being applied.

Effect of Type of Tissue on the Development of Chrysomya rufifacies (Diptera: Calliphoridae) in Sri Lanka.

Chrysomya rufifacies (Macquart), the hairy maggot blow fly, is of great importance for the field of forensic entomology due to its habit as an early colonizer of decomposing vertebrate remains and myiasis producer. Development studies on this species have been conducted in scattered regions of the world, using types of tissue from several species of animals as a rearing medium. Despite the commonality of C. rufifacies in Sri Lanka, developmental studies have never been performed in this region. As well, the effects of diet on development have not been tested. In the current study, C. rufifacies immatures were reared on skeletal muscle, liver, and heart from domestic swine, with flies from colonies maintained at 25 and 28°C. The minimum time needed to complete each stage at 25°C on liver (224.14 h) was fastest followed by skeletal muscle (249.33 h) and heart (251.64 h) respectively, whereas at 28°C, fly development was quickest on heart muscle (178.27 h) followed by liver (178.50 h) and skeletal muscle (186.17 h) respectively. A significant difference in total development time was determined for temperature, while the rearing medium was not significant. Temperature also showed a significant effect on the length and the width of the larvae, while the type of tissue statistically impacted only the width.

Impact of mycolactone produced by Mycobacterium ulcerans on life-history traits of Aedes aegypti (L.) and resulting habitat selection for oviposition.

Buruli ulcer (BU) is a globally recognized, yet largely neglected tropical disease whose etiologic agent is Mycobacterium ulcerans. Although the exact mode of transmission is unclear, epidemiological evidence links BU incidence with slow-moving or stagnant, aquatic habitats, and laboratory-based experiments have shown disease manifestation in animals with dermal punctures. Therefore, hypotheses for transmission include contact with slowmoving aquatic habitats and associated biting aquatic insects, such as mosquitoes. Recent research demonstrated the toxin produced by M. ulcerans, mycolactone, is an attractant for adult mosquitoes seeking a blood-meal as well as oviposition sites. In the study presented here, we examined the impact of mycolactone at different concentrations on immature lifehistory traits of Aedes aegypti, which commonly occurs in the same environment as M. ulcerans. We determined percent egg hatch was not significantly different across treatments. However, concentration impacted the survivorship of larval mosquitoes to the adult stage (p < 0.001). Resulting adults also showed a slight preference, but not significant (p > 0.05), for oviposition in habitats contaminated with mycolactone suggesting a legacy effect.

Field Validation of a Development Data Set for Cochliomyia macellaria (Diptera: Calliphoridae): Estimating Insect Age Based on Development Stage.

Insect age estimates can be useful for estimating the postmortem interval when certain assumptions are met. Such estimates are based on species-specific development data that are temperature-dependent and variable, and therefore prone to different degrees of error depending on the combination of data sets, calculations, and assumptions applied in a specific instance. Because of this potential error, validating the methods employed is necessary for determining accuracy and precision of a given technique. For forensic entomology, validation of development data sets is one approach for identifying the uncertainty associated with insect age estimates. Cochliomyia macellaria (Fabricius) is a primary colonizer of remains across the United States and is commonly encountered in forensic investigations. A development study for this species was produced for a central Texas, U.S. population; the variation associated with this data set and the pre-appearance interval were previously explored in an ecological model. The objectives of this study were to determine the accuracy of the development data and the validity of the ecological model when applied to immatures of known age developing under field conditions. Results indicate this data set is an accurate predictor of insect age when using development stage, supporting the validity of the ecological model in central Texas. Age predictions made with all stages present in a sample were more accurate than predictions made with the most developed stage in a sample, and estimates of age when using the prepupal stage were overestimated regardless of prediction method, though thermal requirements for total development were similar.

Impact of mycolactone produced by Mycobacterium ulcerans on life-history traits of Aedes aegypti (L.) and resulting habitat selection for oviposition

@#Buruli ulcer (BU) is a globally recognized, yet largely neglected tropical disease whose etiologic agent is Mycobacterium ulcerans. Although the exact mode of transmission is unclear, epidemiological evidence links BU incidence with slow-moving or stagnant, aquatic habitats, and laboratory-based experiments have shown disease manifestation in animals with dermal punctures. Therefore, hypotheses for transmission include contact with slowmoving aquatic habitats and associated biting aquatic insects, such as mosquitoes. Recent research demonstrated the toxin produced by M. ulcerans, mycolactone, is an attractant for adult mosquitoes seeking a blood-meal as well as oviposition sites. In the study presented here, we examined the impact of mycolactone at different concentrations on immature lifehistory traits of Aedes aegypti, which commonly occurs in the same environment as M. ulcerans. We determined percent egg hatch was not significantly different across treatments. However, concentration impacted the survivorship of larval mosquitoes to the adult stage (p < 0.001). Resulting adults also showed a slight preference, but not significant (p > 0.05), for oviposition in habitats contaminated with mycolactone suggesting a legacy effect.

Effect of Temperature and Tissue Type on the Development of Myiasis Causing Fly; Chrysomya bezziana (Diptera: Calliphoridae).

Chrysomya bezziana Villeneuve is the major human myiasis producer in tropical countries, including Sri Lanka. Although of great medical and veterinary importance, only a single study has been published on the development of C. bezziana. This limitation is due in part to the difficulty of maintaining this species in a colony outside of using a living host. In this study, a novel technique that overcomes this limitation is presented along with development data for C. bezziana fed on three different meat types: swine muscle, swine liver, and bovine muscle at two temperature regimes: 35 and 37°C. The optimum development of C. bezziana was recorded at 35°C in bovine muscle (262.20 h) followed by swine muscle (286.00 h) and swine liver (307.00 h). Data from the current study indicate tissue type significantly impacts change in length and width of larvae over time, whereas the two temperatures examined had no significant effect.

Mycobacterium ulcerans toxin, mycolactone may enhance host-seeking and oviposition behaviour by Aedes aegypti (L.) (Diptera: Culicidae).

The ecological functions of many toxins continue to remain unknown for those produced by environmental pathogens. Mycobacterium ulcerans, the causative agent of the neglected tropical disease, Buruli ulcer, produces a cytotoxic macrolide, mycolactone, whose function(s) in the environment remains elusive. Through a series of dual-choice behaviour assays, they show that mycolactone may be an interkingdom cue for the yellow fever mosquito, Aedes aegypti, seeking blood-meals as well as oviposition sites. Results provide novel insight into the evolution between bacteria and potential vectors. While further studies are needed to determine if mycolactone is an actual signal rather than simply a cue, this discovery could serve as a model for determining roles for toxins produced by other environmental pathogens and provide opportunities for developing novel strategies for disease prevention. The relationship between M. ulcerans, mycolactone, and Ae. aegypti further suggests there could be an amplification effect for the spread of pathogens responsible for other diseases, such as yellow fever and dengue.

Association of Myianoetus muscarum (Acari: Histiostomatidae) With Synthesiomyia nudiseta (Wulp) (Diptera: Muscidae) on Human Remains.

Synthesiomyia nudiseta (Wulp) (Diptera: Muscidae) was identified during the course of three indoor medicolegal forensic entomology investigations in the state of Texas, one in 2011 from Hayes County, TX, and two in 2015 from Harris County, TX. In all cases, mites were found in association with the sample and subsequently identified as Myianoetus muscarum (L., 1758) (Acariformes: Histiostomatidae). This report represents the first records of a mite associated with S. nudiseta in the continental United States. In particular, this association is believed to be of potential future value in forensic investigations, as it lends new insight into the community structure of colonizers on human remains in indoor environments.

Seasonal and Geographic Variation in Biodiversity of Forensically Important Blow Flies (Diptera: Calliphoridae) in New Jersey, USA.

Determining the time of colonization of human or other animal remains by blow flies (Diptera: Calliphoridae) can play an important role in criminal investigations. However, blow fly presence in a given area is strongly influenced by abiotic and biotic variables such as temperature and habitat. We wanted to assess the biodiversity of adult blow flies in New Jersey, USA, where very little is known about these taxa. Toward that end we collected adult blow flies biweekly from traps baited with bovine liver and placed across three regions in New Jersey over a 2-yr period (2011-2013). We collected and identified 9,257 adult calliphorids, comprising six genera and 12 species. Blow fly assemblages composed of these species varied by season, but community composition did not vary among regions within a given season. Three species, Lucilia coeruleiviridis (Macquart), Lucilia sericata (Meigen), and Phormia regina (Meigen) comprised 88.5% of all adult blow flies collected (42.6, 25.9, 20.0%, respectively). Combining all regions, the dominant species for both spring and summer was L. coeruleiviridis comprising 35.1% of all adults caught in spring and 64.1% in summer. P. regina was the dominant species in fall, totaling 40.1% of all adults caught and Calliphora vicina (Robineau-Desvoidy) was the dominant species for winter, totaling 44.8% of all adults caught. Our findings provide the first assessment of blow fly communities in New Jersey, and these results can be applied to surrounding states where data are severely lacking for forensic application.

First record of Megaselia scalaris (LOEW) (DIPTERA: PHORIDAE) infesting a spinose ear tick, Otobius megnini, colony in Sri Lanka.

Megaselia scalaris (Loew) is a cosmopolitan polyphagous small fly with the ability of exploiting variety of ecological niches. Different life history stages act as detritivore, parasite, and parasitoid of wider spectrum of plant and animal matter under natural and laboratory conditions. Here, for the first time we present the opportunistic parasitism of M. scalaris on Otobius megnini, which act as a vector of Q fever and is capable of causing paralysis, toxic conditions, otoacariasis and otitis in humans and other animals. Tick samples from the ear canals of 14 thoroughbred horses were brought to the laboratory and several days later, larvae of M. scalaris were found feeding on immature stages of O. megnini. When the development was completed pupae were found attached to adult ticks and all nymphs were found dead. This context reveals the capability of M. scalaris surviving on O. megnini and the risk of their invading ear canals of horses.

First record of Megaselia scalaris (LOEW) (DIPTERA: PHORIDAE) infesting a spinose ear tick, Otobius megnini, colony in Sri Lanka

Megaselia scalaris (Loew) is a cosmopolitan polyphagous small fly with the ability of exploiting variety of ecological niches. Different life history stages act as detritivore, parasite, and parasitoid of wider spectrum of plant and animal matter under natural and laboratory conditions. Here, for the first time we present the opportunistic parasitism of M. scalaris on Otobius megnini, which act as a vector of Q fever and is capable of causing paralysis, toxic conditions, otoacariasis and otitis in humans and other animals. Tick samples from the ear canals of 14 thoroughbred horses were brought to the laboratory and several days later, larvae of M. scalaris were found feeding on immature stages of O. megnini. When the development was completed pupae were found attached to adult ticks and all nymphs were found dead. This context reveals the capability of M. scalaris surviving on O. megnini and the risk of their invading ear canals of horses.

Morphology of puparia of flesh flies in Thailand.

Puparia of five flesh fly species were investigated for forensic study. Boettcherisca nathani (Lopes, 1961), Boettcherisca peregrina (Robineau-Desvoidy, 1830), Lioproctia pattoni (Senior-White, 1924), Liopygia ruficornis (Fabricius, 1794) and Parasarcophaga (Liosarcophaga) dux (Thomson, 1869) were examined with a scanning electron microscopy (SEM). Differences between species were found in the number and arrangement of papillae in the anterior spiracle, the shape of intersegmental spines between the prothorax and mesothorax and the pattern of spiracular tufts at the posterior spiracle. The anterior spiracle of B. nathani had two rows, comprising 21-27 papillae; while those of B. peregrina and L. pattoni had one or two irregular rows with 24-26 and 20-28 papillae, respectively. Anterior spiracle of L. ruficornis and P. dux had one row of 10-15 papillae. Intersegmental spines between the prothorax and mesothorax and pattern of spiracular tufts at the posterior spiracle are morphologically different. L. ruficornis and P. dux puparia are similar, but the position of the interslit plate between the inner and middle spiracular slits was found to be an important attribute to separate both species. Morphometric analysis on the length and width of puparia of these species revealed statistically different among them. The key for identifying puparia of forensically important flesh flies has been provided.

Ontogenensis and developmental rate of the blow fly, Hypopygiopsis tumrasvini Kurahashi (Diptera: Calliphoridae).

Blow flies of the genus Hypopygiopsis are considered forensically important. In Thailand, four Hypopygiopsis species coexist, i.e., Hypopygiopsis fumipennis, Hypopygiopsis infumata, Hypopygiopsis violacea and Hypopygiopsis tumrasvini. In this study, the ontogeny and developmental rate of H. tumrasvini eggs, larvae and pupae were determined in the laboratory chamber reared at 25.0 ± 2.0°C and 80.0 ± 5.0% RH. Larvae emerged from eggs 10-12 h after deposition. Mean length of the first, second, third (feeding phase), third (post-feeding phase) instars and puparia were 3.5 ± 1.1, 7.2 ± 1.1, 13.5 ± 1.8, 12.5 ± 0.5 and 9.0 ± 0.7 mm, respectively. The median development time for first, second, third instar (feeding phase), third instar (post-feeding phase) and pupariation period was 8 h, 10 h, 34 h, 22 d and 9-10 d, respectively. Developmental curve of the larval length indicated the rapid progression from 0 until 40 h from the first instar until the feeding third instar. Video recording of pupariation revealed the development of pupal respiratory horn beneath the larval integument at 27.0 h; whereas it protruded through the orifice of the integument at 27.5 h.

Substrate effects on pupation and adult emergence of Hermetia illucens (Diptera: Stratiomyidae).

Black soldier flies, Hermetia illucens (L.) (Diptera: Stratiomyidae), are of particular interest for their applications in waste management. Feeding on decaying organic waste, black soldier flies successfully reduce manure in confined animal feeding operations of poultry, swine, and cattle. To optimize waste conversion in confined animal feeding operations and landfill facilities, it is imperative to optimize black soldier fly development. Unfortunately, black soldier flies only convert waste during their larval feeding stages and therefore it is of interest to optimize the nonfeeding stages of development, specifically, the postfeeding and pupal stages. The time spent in these stages is thought to be determined by the pupation substrate encountered by the postfeeding larvae. The objective of this study was to determine the effect different pupation substrates have on postfeeding development time, pupation time, and adult emergence success. Five pupation substrates were compared: wood shavings, potting soil, topsoil, sand, and nothing. Postfeeding larvae took longer to reach pupation in the absence of a pupation substrate, although reaching pupation in the shortest time in potting soil and wood shavings. The time spent in the pupal stage was shortest in the absence of a pupation substrate. However, fewer adults emerged when a pupation substrate was not provided.

Seasonal necrophagous insect community assembly during vertebrate carrion decomposition.

Necrophagous invertebrates have been documented to be a predominant driver of vertebrate carrion decomposition; however, very little is understood about the assembly of these communities both within and among seasons. The objective of this study was to evaluate the seasonal differences in insect taxa composition, richness, and diversity on carrion over decomposition with the intention that such data will be useful for refining error estimates in forensic entomology. Sus scrofa (L.) carcasses (n = 3-6, depending on season) were placed in a forested habitat near Xenia, OH, during spring, summer, autumn, and winter. Taxon richness varied substantially among seasons but was generally lower (1-2 taxa) during early decomposition and increased (3-8 taxa) through intermediate stages of decomposition. Autumn and winter showed the highest richness during late decomposition. Overall, taxon richness was higher during active decay for all seasons. While invertebrate community composition was generally consistent among seasons, the relative abundance of five taxa significantly differed across seasons, demonstrating different source communities for colonization depending on the time of year. There were significantly distinct necrophagous insect communities for each stage of decomposition, and between summer and autumn and summer and winter, but the communities were similar between autumn and winter. Calliphoridae represented significant indicator taxa for summer and autumn but replaced by Coleoptera during winter. Here we demonstrated substantial variability in necrophagous communities and assembly on carrion over decomposition and among seasons. Recognizing this variation has important consequences for forensic entomology and future efforts to provide error rates for estimates of the postmortem interval using arthropod succession data as evidence during criminal investigations.

Transient gut retention and persistence of Salmonella through metamorphosis in the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae).

AIMS: This study was undertaken to determine the retention of Salmonella through Alphitobius diaperinus metamorphosis and its contribution, through defecation, to external contamination. METHODS AND RESULTS: Insects were exposed to a tagged Salmonella enterica and evaluated for external elimination. (i) Each day for 3 weeks, a filter collected frass from a restrained insect for analysis. (ii) Exposed larvae in a closed container were followed through pupation, and newly emerged adults were examined for their retention of marker bacteria. CONCLUSIONS: Exposed adults and larvae produced Salmonella-positive frass for an average of 8 days, ranging from 6 to 11 days and 6 to 12 days, respectively. Nineteen per cent of the larvae carried Salmonella through metamorphosis and eclosion, with 5% of the pupal exuviae being positive as well. SIGNIFICANCE AND IMPACT OF THE STUDY: Many sources of foodborne pathogens within the poultry production facilities, including reservoir populations, currently go unrecognized. This diminishes the ability of producers to mitigate the transfer of pathogens between animals, humans and the environment. Poultry management standards accept the reutilization of litter. Alphitobius diaperinus survive between flock rotations on the reutilized litter, and it was demonstrated in this study that the Salmonella they carry can survive with them.

A roadmap for bridging basic and applied research in forensic entomology.

The National Research Council issued a report in 2009 that heavily criticized the forensic sciences. The report made several recommendations that if addressed would allow the forensic sciences to develop a stronger scientific foundation. We suggest a roadmap for decomposition ecology and forensic entomology hinging on a framework built on basic research concepts in ecology, evolution, and genetics. Unifying both basic and applied research fields under a common umbrella of terminology and structure would facilitate communication in the field and the production of scientific results. It would also help to identify novel research areas leading to a better understanding of principal underpinnings governing ecosystem structure, function, and evolution while increasing the accuracy of and ability to interpret entomological evidence collected from crime scenes. By following the proposed roadmap, a bridge can be built between basic and applied decomposition ecology research, culminating in science that could withstand the rigors of emerging legal and cultural expectations.

Seasonal colonization and decomposition of rat carrion in water and on land in an open field in South Carolina.

Decomposition and insect colonization of rat, Rattus rattus L., carrion on land and in water were compared during summer and winter in a plowed field in northwestern South Carolina. During winter, carcasses on land reached the dried-remains stage of decomposition, whereas carcasses in water reached the early-floating stage. During summer, carcasses in both habitats entered the final-remains stage of decomposition in 1-2 wk. Fewer than 30 species of carrion insects were recorded from the carcasses over the duration of the study, probably reflecting the small size of the carcasses and the depauperate fauna of the habitat. Three species of blow flies--Cynomyopsis cadaverina (Robineau-Desvoidy), Calliphora vicina Robineau-Desvoidy, and Lucilia illustris (Meigen)--colonized carrion on land during winter, but no insects colonized carrion in water during winter. Two species of blow flies, Cochliomyia macellaria (F.) and Phaenicia sericata (Meigen), and 1 species of flesh fly, Sarcophaga bullata Parker, colonized the carrion on land and in water during summer; the blow fly, Phormia regina (Meigen), colonized only the carrion on land. This study demonstrated seasonal variation in decomposition and colonization patterns of carrion in contrasting habitats, with important implications for forensic entomology.

Intraperitoneal cisplatin and regional hyperthermia for ovarian carcinoma.

PURPOSE: To review the theoretical basis and results of a Phase I study of concurrent intraperitoneal cisplatin and hyperthermia in the treatment of ovarian carcinoma. METHODS AND MATERIALS: Previously treated patients with epithelial ovarian carcinoma received intraperitoneal instillation of cisplatin and 60 minutes of regional hyperthermia, with a goal temperature of 41.5 degrees C. Cisplatin dose started at 20 mg/m2 with escalation to the maximally tolerated dose. Six such cycles given every 3 weeks were planned. Pharmacokinetic studies with and without hyperthermia were performed. RESULTS: Fifteen patients receiving 17 courses of treatment were evaluable. The maximally tolerated dose of cisplatin was between 80 and 120 mg/m2. The dose limiting toxicity was nephrotoxicity in all but one course. The median intraperitoneal temperature was 40.7 degrees C; the majority of treatments in which the goal temperature was not reached had power limited by patient discomfort. No major toxicities attributable to hyperthermia were noted. Pharmacokinetic studies noted no significant differences between treatments with vs. without hyperthermia, with intraperitoneal to plasma area under the curve ratios being 30-35. Ten patients had a decline in their CA-125 count during treatment, although in only two patients did this response persist beyond their course of treatment. CONCLUSION: Intraperitoneal cisplatin and regional hyperthermia can be performed with reasonable toxicity. The maximally tolerated dose of 80-120 mg/m2 in pretreated patients (which is similar to those reported with cisplatin alone) and median intraperitoneal temperatures of 40.7 degrees C, however, are felt to be too low to be efficacious in a significant percentage of women with bulky recurrent disease. Further study using intravenous thiosulfate and controlled analgesia is being performed.