Baneshwar Singh

A Survey of Bacterial Diversity From Successive Life Stages of Black Soldier Fly (Diptera: Stratiomyidae) by Using 16S rDNA Pyrosequencing

ABSTRACT Sustainable methods for managing waste associated with people and animals have been proposed in the past. Black soldier fly, Hermetia illucens (L.), larvae represent one of the more promising methods. Larvae reduce dry matter, bacteria, offensive odor, and house fly populations. Prepupae can be used as feedstuff for livestock. However, it is not known if such a method results in the proliferation of potential pathogens. Although some bacterial species have been cultured and identified from black soldier fly, a true appreciation of fly associated bacterial diversity is not known. Such information is needed to understand pathogen colonization on decomposing animal and plant waste in the presence of black soldier fly larvae as well as develop research strategies for maximizing the use of this fly to reduce waste without risking environmental harm. Using 454 sequencing, we surveyed bacterial diversity associated with successive life stages of the black soldier fly reared on plant material. Bacteria diversity classified (99.8%) across all life stages spanned six bacterial phyla with >80% bootstrap support. Bacteroidetes and Proteobacteria were the most dominant phyla associated with the black soldier fly accounting for two-thirds of the fauna identified. Many of these bacteria would go undetected because of their inability to be cultured.

Molecular Systematics of the Calliphoridae (Diptera: Oestroidea): Evidence From One Mitochondrial and Three Nuclear Genes

ABSTRACT Approximately 8% of calyptrate species diversity comes from the Calliphoridae, which includes flies of medical, veterinary, and forensic importance. The status of family Calliphoridae has for years been the central systematic problem of the superfamily Oestroidea, and phylogenetic relationships between the key groups of the Calliphoridae are unresolved and controversial. We reconstructed phylogenies of the Calliphoridae within the larger context of the other Oestroidea based on 5,189 bp of combined data from one mitochondrial (cytochrome oxidase subunit one) and three nuclear (carbamoylphosphate synthetase, elongation factor one alpha, and 28S ribosomal RNA) genes using maximum parsimony, maximum likelihood, and Bayesian methods. Trees obtained from the different phylogenetic methods were almost identical. Calliphoridae is polyphyletic, with the phylogenetic position of Mesembrinellinae still uncertain but clearly outside the lineage that includes other Calliphoridae and some noncalliphorids, and Polleniinae is the sister group of the family Tachinidae. Strong support for a sister group relationship between Rhiniinae and traditional calliphorid subfamilies conflicts with a recent proposal to give Rhiniinae family status. All calliphorid subfamilies (except Calliphorinae) for which we had more than one species were monophyletic. Melanomyinae was nested within Calliphorinae. Toxotarsinae was more closely related to Calliphorinae rather than, as indicated by morphology, to Chrysomyinae. Efforts to resolve the relationships of the Oestroid families were largely inconclusive, although the monophyly of the superfamily was strongly supported.

Bacteria Mediate Oviposition by the Black Soldier Fly, Hermetia illucens (L.), (Diptera: Stratiomyidae)

There can be substantial negative consequences for insects colonizing a resource in the presence of competitors. We hypothesized that bacteria, associated with an oviposition resource and the insect eggs deposited on that resource, serve as a mechanism regulating subsequent insect attraction, colonization, and potentially succession of insect species. We isolated and identified bacterial species associated with insects associated with vertebrate carrion and used these bacteria to measure their influence on the oviposition preference of adult black soldier flies which utilizes animal carcasses and is an important species in waste management and forensics. We also ascertained that utilizing a mixture of bacteria, rather than a single species, differentially influenced behavioral responses of the flies, as did bacterial concentration and the species of fly from which the bacteria originated. These studies provide insight into interkingdom interactions commonly occurring during decomposition, but not commonly studied.

A Review of Bacterial Interactions With Blow Flies (Diptera: Calliphoridae) of Medical, Veterinary, and Forensic Importance

Abstract Blow flies are commonly associated with decomposing material. In most cases, the larvae are found feeding on decomposing vertebrate remains; however, some species have specialized to feed on living tissue or can survive on other alternate resources like feces. Because of their affiliation with such septic environments, these insects have close associations with microbes. Historically, a tremendous amount of research focused on these insects due to their veterinary importance. Within the past 40 yr, efforts have expanded this research to include areas such as systems ecology, forensics, and even wound debridement (maggot) therapy. Initial research efforts examining the relationship between microbes and these insects were hampered by the technology available. However, with the advent of high-throughput sequencing and modern molecular techniques, new avenues of research examining these interactions have opened up. The purpose of this article is to highlight the research exploring the interactions between microbes and blow flies with regards to blow fly biology, the application of such information to benefit humanity, and potential future pathways of research.

Field Documentation of Unusual Post-Mortem Arthropod Activity on Human Remains.

ABSTRACT During a forensic investigation, the presence of physical marks on human remains can influence the interpretation of events related to the death of an individual. Some tissue injury on human remains can be misinterpreted as ante- or peri-mortem wounds by an investigator when in reality the markings resulted from post-mortem arthropod activity. Unusual entomological data were collected during a study examining the decomposition of a set of human remains in San Marcos, Texas. An adult female Pediodectes haldemani (Girard) (Orthoptera: Tettigoniidae) and an Armadillidium cf. vulgare (Isopoda: Armadilidiidae) were documented feeding on the remains. Both arthropods produced physical marks or artifacts on the remains that could be misinterpreted as attack, abuse, neglect, or torture. Additionally, red imported fire ants, Solenopsis invicta Buren (Hymenoptera: Formicidae), were observed constructing structures in the mark produced by the P. haldemani feeding. These observations provide insight into the potential of post-mortem arthropod damage to human remains, which previously had not been described for these taxa, and therefore, physical artifacts on any remains found in similar circumstances may result from arthropod activity and not ante- or peri-mortem wounds.

DNA-Based Identification of Forensically Important Blow Flies (Diptera: Calliphoridae) From India

Abstract Correct species identification is the first and the most important criteria in entomological evidence-based postmortem interval (PMI) estimation. Although morphological keys are available for species identification of adult blow flies, keys for immature stages are either lacking or are incomplete. In this study, cytochrome oxidase subunit 1 (COI) reference data were developed from nine species (belonging to three subfamilies, namely, Calliphorinae, Luciliinae, and Chrysomyinae) of blow flies from India. Seven of the nine species included in this study were found suitable for DNA-based identification using COI gene, because they showed nonoverlapping intra- (0.0–0.3%) and inter-(1.96–18.14%) specific diversity, and formed well-supported monophyletic clade in phylogenetic analysis. The remaining two species (i.e., Chrysomya megacephala (Fabricius) and Chrysomya chani Kurahashi) cannot be distinguished reliably using our database because they had a very low interspecific diversity (0.11%), and Ch. megacephala was paraphyletic with respect to Ch. chani in the phylogenetic analysis. We conclude that the COI gene is a useful marker for DNA-based identification of blow flies from India.

Interkingdom Cues by Bacteria Associated with Conspecific and Heterospecific Eggs of Cochliomyia macellaria and Chrysomya rufifacies (Diptera: Calliphoridae) Potentially Govern Succession on Carrion

Abstract Deciphering mechanisms that regulate succession on ephemeral resources is critical for elucidating food web dynamics and nutrient recycling. Blow fly (Diptera: Calliphoridae) colonization and utilization of vertebrate carrion serve as a model for such studies, as they are the primary invertebrates that recycle this ephemeral resource. Initial colonization by blow flies often results in heightened attraction and colonization by competing conspecifics and heterospecifics, thereby regulating associated arthropod succession patterns. We examined the response of Cochliomyia macellaria (F.) and Chrysomya rufifacies (Macquart) to conspecific and heterospecific eggs. Because Ch. rufifacies is facultatively predacious and cannabalistic, we hypothesized that adults would recognize the presence of conspecific and heterospecific eggs, thus avoiding potential predation and competition. Using a Y-tube olfactometer, we measured the residence time response of C. macellaria and Ch. rufifacies to conspecific and heterospecific eggs of three different age classes (fresh to 9-h-old). Fly responses to surface-sterilized eggs and to an aqueous solution containing egg-associated microbes were then examined. High-throughput sequencing was used to survey egg-associated bacteria from both species. We report that C. macellaria and Ch. rufifacies exhibit differential responses to eggs of conspecifics and heterospecifics, which appear to be a result of microbial volatile-related odors. These behaviors likely influence predator–prey interactions between species. Preliminary high-throughput sequencing revealed Ch. rufifacies had a similar egg-associated fauna as C. macellaria, which may serve as a form of camouflage, allowing it to colonize and thereby attract C. macellaria, a common prey for its larvae.

Temporal and Spatial Impact of Human Cadaver Decomposition on Soil Bacterial and Arthropod Community Structure and Function

As vertebrate carrion decomposes, there is a release of nutrient-rich fluids into the underlying soil, which can impact associated biological community structure and function. How these changes alter soil biogeochemical cycles is relatively unknown and may prove useful in the identification of carrion decomposition islands that have long lasting, focal ecological effects. This study investigated the spatial (0, 1, and 5 m) and temporal (3–732 days) dynamics of human cadaver decomposition on soil bacterial and arthropod community structure and microbial function. We observed strong evidence of a predictable response to cadaver decomposition that varies over space for soil bacterial and arthropod community structure, carbon (C) mineralization and microbial substrate utilization patterns. In the presence of a cadaver (i.e., 0 m samples), the relative abundance of Bacteroidetes and Firmicutes was greater, while the relative abundance of Acidobacteria, Chloroflexi, Gemmatimonadetes, and Verrucomicrobia was lower when compared to samples at 1 and 5 m. Micro-arthropods were more abundant (15 to 17-fold) in soils collected at 0 m compared to either 1 or 5 m, but overall, micro-arthropod community composition was unrelated to either bacterial community composition or function. Bacterial community structure and microbial function also exhibited temporal relationships, whereas arthropod community structure did not. Cumulative precipitation was more effective in predicting temporal variations in bacterial abundance and microbial activity than accumulated degree days. In the presence of the cadaver (i.e., 0 m samples), the relative abundance of Actinobacteria increased significantly with cumulative precipitation. Furthermore, soil bacterial communities and C mineralization were sensitive to the introduction of human cadavers as they diverged from baseline levels and did not recover completely in approximately 2 years. These data are valuable for understanding ecosystem function surrounding carrion decomposition islands and can be applicable to environmental bio-monitoring and forensic sciences.

Evaluation of DNA Extraction Methods from Waterlogged Bones: A Pilot Study,

When deaths occur in water, soft tissue decomposes after a temperature‐dependent period, making DNA identification dependent on bone. This study examined the effects of water on bone DNA purity and quantity, and determined the best of three extraction methods for isolating DNA. The organic phenol–chloroform method consistently extracted DNA approximating the accepted 260/280 purity value (~1.8); ChargeSwitch® gDNA Plant Kit and DNeasy Blood and Tissue Kit produced fair and unacceptable values, respectively. The purity value for humerus and rib samples was consistent across accumulated degree days (ADD). Significant differences in quantification among extraction methods and between bone types were identified. Ribs and ChargeSwitch® gDNA Plant Kit samples produced the lowest mean Ct values of the bone types and the extraction methods, respectively. Therefore, this study proposes that magnetic bead technology extraction methods and ribs be considered when processing bones that have been submerged in water for any length of time.

An accurate bacterial DNA quantification assay for HTS library preparation of human biological samples

Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on quantity of total DNA isolated. This method can miscalculate bacterial DNA quantity due to the mixed nature of the sample, and consequently library preparation is often unreliable. We developed an assay that can accurately and specifically quantify bacterial DNA within a mixed sample for reliable 16S ribosomal DNA (16S rDNA) library preparation and high throughput sequencing (HTS). A qPCR method was optimized using universal 16S rDNA primers, and a commercially available bacterial community DNA standard was used to develop a precise standard curve. Following qPCR optimization, 16S rDNA libraries from saliva, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS data were generated with as low as 20 pg of bacterial DNA. Changes in bacterial DNA quantity did not impact observed relative abundances of major bacterial taxa, but relative abundance changes of minor taxa were observed. Accurate quantification of microbial DNA resulted in consistent, successful library preparations for HTS analysis.