50 years later, insect evidence overturns Canada's most notorious case - Regina v. Steven Truscott.

On 11 June 1959, the body of 12-year old Lynne Harper was discovered in a woodlot northeast of Clinton, Ontario. Although insect evidence was photographed and collected at the scene and autopsy, this evidence was not used in the 1959 trial. Instead, time of death was pinpointed to a 45-min window of 1900-1945 h on 9 June 1959 based on stomach content analysis. Based on circumstantial evidence and this time frame that he was the last suspect to see her alive, 14-year old Steven Truscott was convicted of her murder. He was scheduled to be hanged, but a temporary reprieve postponed his execution. In 1960, his death sentence was commuted to life imprisonment. Truscott was the youngest person to be sentenced to death in Canada, and his case provided the major impetus toward abolition of the death penalty in Canada. Truscott always maintained his innocence. In 2001, the Association in Defence of the Wrongly Convicted filed an appeal to have the case reopened. In 2006, the authors of this paper were contacted by Attorneys James Lockyer and Phil Campbell of the LCP Law Firm in Toronto to investigate this case. Fresh evidence was presented at the Ontario Court of Appeal in 2006-2007 including testimony of 3 forensic entomologists. This resulted in controversy regarding identification of the insects and assumptions of insect behaviour that affected the postmortem interval estimate. Lack of scientific evidence for the controversial theories proposed by one testifying entomologist resulted in disregarding his testimony. Instead, testimony by VanLaerhoven and Merritt was accepted. Based on their analysis and a re-creation experiment of the insect evidence, initial fly colonization occurred during daylight hours of 10 June 1959. The collected larvae were not likely to have been deposited on the body before dark (2140 h) on 9 June 1959 as this would have resulted in significantly larger and more advanced larval instar than were collected at the scene or autopsy. This analysis, together with a pathology reanalysis of stomach content analysis, demonstrated that the original estimate of time of death was unreliable. Truscott was with numerous witnesses prior to 1900 h and after 2000 h on 9 June 1959, thus the estimate of time of death was the most critical evidence in the original 1959 trial and the 2006-2007 appeal. On 28 August 2007, his conviction was overturned, declared a wrongful conviction and miscarriage of justice. Steven Truscott was acquitted of the murder charges.

Insect arrival pattern and succession on buried carrion in Michigan.

This study examined pig carcasses buried at two different depths, 30 and 60 cm, to determine if insects were able to colonize buried carcasses, when they arrive at each depth, and what fauna were present over seven sampling dates to establish an insect succession database on buried carrion in East Lansing, MI. Thirty-eight pigs were buried, 18 at 30 cm and 20 at 60 cm. Four control carcasses were placed on the soil surface. Three replicates at each depth were exhumed after 3, 7, 14, 21, 30, and 60 d, respectively. One pig also was exhumed from 60 cm after 90 d and another after 120 d. Sarcophaga bullata (Parker) (Diptera: Sarcophagidae) and Hydrotaea sp. (Diptera: Muscidae) were found colonizing buried carrion 5 d after burial at 30 cm. Insect succession at 30 cm proceeded with flesh and muscid flies being the first to colonize, followed by blow flies. Insects were able to colonize carcasses at 60 cm and Hydrotaea sp. and Megaselia scalaris (Loew) (Diptera: Phoridae) were collected 7 d after burial. Insect succession at 60 cm did not proceed similarly, instead muscid and coffin flies were the only larvae collected. Overall these results reveal postburial interval estimates for forensic investigations in mid-Michigan during the summer, depending on climatic and soil conditions.

An acute trophic cascade among microorganisms in the tree hole ecosystem following removal of omnivorous mosquito larvae.

Results of two field experiments showed that selective removal of omnivorous mosquito larvae (Aedes triseriatus (Say)) functioning as top predators in the food web of a temperate, tree hole ecosystem resulted rapidly in increased abundance of flagellate and then ciliate populations. Flagellate density increased from <1 per ml to >103 per ml within 4 days of omnivore removal, followed shortly thereafter by an increase in ciliate density from <1 per ml to >102 per ml, after which flagellate density declined, and flagellate and ciliate densities stabilized. Rod-shaped bacteria increased slightly in density after removal of larval mosquitoes, then declined as protist density increased. Cocciform bacteria did not vary in density with these changes, thus the trophic cascade dampened at the remotest trophic level. Concomitant with the increase in protist densities, some bacteria formed elongated filaments >10 µm in length, likely an anti-predation, morphological response stimulated by suddenly intensified grazing as protozoan density rose. Results suggest that feeding by omnivorous mosquito larvae exhibited strong top-down effects on flagellate and ciliate populations, depressing them to below their equilibrium densities and nearly to extinction in tree hole ecosystems.

Field and laboratory investigations on the effects of road salt (NaCl) on stream macroinvertebrate communities.

Field and laboratory experiments were conducted to examine the effects of road salt (NaCI) on stream macroinvertebrates. Field studies investigated leaf litter processing rates and functional feeding group composition at locations upstream and downstream from point source salt inputs in two Michigan, USA streams. Laboratory studies determined the effects of increasing NaCl concentrations on aquatic invertebrate drift, behavior, and survival. Field studies revealed that leaves were processed faster at upstream reference sites than at locations downstream from road salt point source inputs. However, it was sediment loading that resulted in partial or complete burial of leaf packs, that affected invertebrate activity and confounded normal leaf pack colonization. There were no significant differences that could be attributed to road salt between upstream and downstream locations in the diversity and composition of invertebrate functional feeding groups. Laboratory drift and acute exposure studies demonstrated that drift of Gammarus (Amphipoda) may be affected by NaCl at concentrations greater than 5000 mg/l for a 24-h period. This amphipod and two species of limnephilid caddisflies exhibited a dose response to salt treatments with 96-h LC50 values of 7700 and 3526 mg NaCl/l, respectively. Most other invertebrate species and individuals were unaffected by NaCl concentrations up to 10,000 mg/l for 24 and 96 h, respectively.

Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) established in the vicinity of Knoxville, Tennessee, USA.

The hairy maggot blow fly, Chrsomya rufifacies (Macquart) (Diptera: Calliphoridae) was collected in large numbers as both adults and immatures in the Knoxville, Tennessee, area during 1998 and is likely established there. The distribution of this species in the Old World, isothermal data, and its collection from mid-Michigan during 1998 suggest that it will eventually occupy most of the U.S. The forensic importance of C. rufifacies makes it probable that it will factor into an increasing number of medicolegal cases, but the expanding distribution of this species decreases its utility as a geographic indicator when postmortem movement of decedents is suspected.

Effects of larval mosquitoes (Aedes triseriatus) and stemflow on microbial community dynamics in container habitats.

The dynamics of the microbial food sources for Aedes triseriatus larvae in microcosms were found to be strongly influenced by larval presence. The total abundance of bacteria in water samples generally increased in response to larvae, including populations of cultivable, facultatively anaerobic bacteria. Additionally, a portion of the community shifted from Pseudomonaceae to Enterobacteriaceae. Bacterial abundance on leaf material was significantly reduced in the presence of actively feeding larvae. Principle-component analysis of whole community fatty acid methyl ester (FAME) profiles showed that larvae changed the microbial community structure in both the water column and the leaf material. Cyclopropyl FAMEs, typically associated with bacteria, were reduced in microcosms containing larvae; however, other bacterial fatty acids showed no consistent response. Long-chain polyunsaturated fatty acids characteristic of microeukaryotes (protozoans and meiofauna) declined in abundance when larvae were present, indicating that larval feeding reduced the densities of these microorganisms. However, presumed fungal lipid markers either increased or were unchanged in response to larvae. Larval presence also affected microbial nitrogen metabolism through modification of the physiochemical conditions or by grazing on populations of bacteria involved in nitrification-denitrification. Stemflow primarily influenced inorganic ion and organic compound concentrations in the microcosms and had less-pronounced effects on microbial community parameters than did larval presence. Stemflow treatments diluted concentrations of all inorganic ions (chloride, sulfate, and ammonium) and organic compounds (total dissolved organic carbon, soluble carbohydrates, and total protein) measured, with the exceptions of nitrite and nitrate. Stemflow addition did not measurably affect larval biomass in the microcosms but did enhance development rates and early emergence patterns of adults.

New strategies for the control of the parthenogenetic chironomid (Paratanytarsus grimmii) (Diptera:Chironomidae) infecting water systems.

Control of the midge, Paratanytarsus grimmii, infesting municipal water systems has proven to be difficult, because it is a parthenogenetic species that can oviposit as a pharate adult and reproduce within the system. Mean densities of P. grimmii in a midwestern USA water distribution system ranged from approximately 140 to 560 individuals/sampling date, and all 4 instars and pupae were present throughout the sampling period. Two products were tested as potential chemical controls: Cat-Floc LS, a coagulant produced by the Calgon Corporation, and 35% hydrogen peroxide, a water purifier. The results of laboratory bioassays showed that Cat-Floc LS over a 15-day period was most effective against P. grimmii.

Growth of Anopheles mosquito larvae on dietary microbiota in aquatic surface microlayers.

Hydrophobic organic matter accumulates under the surface film of water bodies to form the surface microlayers. Heterotrophic microorganisms use this organic matter for growth, and they, in turn, are fed upon by Anopheles mosquito larvae and other animals. From laboratory experiments we show that two species of mosquito larvae, Anopheles gambiae and An.quadrimaculatus, grew most rapidly where surface microlayers were present and, especially, where labile dissolved organic matter was added to promote growth of microorganisms. The importance of microorganisms was confirmed by the addition of gentamicin antibiotic, which suppressed the microbiota and reduced the growth of larvae feeding on surface microlayers. Anopheles larvae grew well on a suspension of finely ground fish food to which the antibiotic had been added, showing that reduced growth was not due to gentamicin itself. Because sub-surface microorganisms are the components of the larval diet that most affect growth, we discuss their relevance to strategies for larval control of Anopheles mosquitoes.

Effects of episodic sedimentation on the net-spinning caddisflies Hydropsyche betteni and Ceratopsyche sparna (Trichoptera: Hydropsychidae).

Net-spinning caddisflies commonly inhabit the chronically sedimented waters of midwestern agricultural catchments despite the presumed costs of sedimentation-induced gill-and net-fouling. We conducted a laboratory experiment to determine whether larval growth and survival of Hydropsyche betteni and Ceratopsyche sparna are affected by daily exposure to moderate levels of sedimentation. Sedimentation did not alter the relative growth rate of either species although slight losses by H. betteni and gains by C. sparna produced significant differences between species. H. betteni had a decreased likelihood of survival in sedimentation treatments relative to controls, but significantly outperformed C. sparna overall. Larval C. sparna are reportedly more sensitive than H. betteni to the instream effects of agricultural land use; however, the results of this experiment suggest that this differential sensitivity is probably not caused by episodic increases in suspended sediment concentration.

Bacterial enrichment in the surface microlayer of an Anopheles quadrimaculatus (diptera: culicidae) larval habitat.

Sampling of the surface microlayer of water in a marsh habitat of Anopheles quadrimaculatus Say in Michigan revealed it to be enriched with bacteria compared with subsurface water samples. Concentrations of total numbers of bacteria ranged from 18.8 x 10(6)/ml to 65.4 x 10(6)/ml (mean, 35.9 x 10(6)/ml; n = 13) in surface microlayer samples, and from 3.8 x 10(6)/ml to 14.3 x 10(6)/ml, (mean, 7.8 x 10(6)/ml; n = 13) in subsurface samples. Specifically, the surface microlayer had higher concentrations of three morphotypes of bacteria (cocci, rods, and rods attached to detritus particles). Given that bacteria are an important food of mosquito larvae, we suggest that the interfacial feeding behavior of An. quadrimaculatus larvae allows them to exploit the surface microlayer, a food-rich zone.

Behavior of larval Aedes triseriatus (Diptera: Culicidae).

The behavior of Aedes triseriatus (Say) fourth instars was studied in laboratory microcosms. A list of 14 larval behaviors was developed from observations of 108 larvae performing 1,836 different acts, and a time budget was developed from 88 larvae by recording the proportion of time each larva spent in the different behavioral states. Larval behavior was organized into clusters of surface and subsurface activities, linked by "dive" and "rise" behaviors. Eight behaviors formed the major activity pattern for larvae and dominated the time budget. Feeding behavior consisted of suspension feeding, feeding at the air-water interface, brushing container and leaf surfaces, and chewing leaf veins. Most (90.8%) of the time budget was devoted to feeding, and larvae spent 52.5% of their time feeding at or near the surface of the water and 37.5% brushing while submerged. When feeding at the air-water interface, larvae rotated about the axis formed by the respiratory siphon in alternating clockwise and counterclockwise directions, possibly to rest the muscles used to twist the larval body into the U-shaped posture characterizing this feeding behavior. Our studies showed that Ae. triseriatus larvae were not exclusively brushers nor bottom feeders as has been assumed for many Aedes larvae but showed great flexibility in feeding, where nearly the entire habitat was the "feeding zone."

Natural food and feeding behavior of Coquillettidia perturbans larvae.

The natural particulate food of larval Coquillettidia perturbans was studied through gut analysis using staining with 4'6-diamidino-2-phenylindole and epifluorescence microscopy. Bacteria (cocci, rods, spirochetes, purple bacteria and cyanobacteria), detritus, euglenoid protozoans and algae (desmids and diatoms) comprised the majority of particulate food, in order of abundance; other protozoans were rare, and hyphal forms (actinomycetes and fungi) were not observed. Abundance of food items in guts did not vary greatly among sampling months (June-December, 1987) or sampling sites. Fourth instars had a greater proportion of euglenoids and algae in their food than did younger instars. Observation of larval feeding behavior showed that fourth instars oriented upside down and fed mainly by suspension feeding, with occasional brushing from sediments and root surfaces. Microtrichia on filaments of the lateral palatal brushes of fourth instars indicated that the larvae were adapted to collect fine particles.

Sampling, seasonal abundance, and mermithid parasitism of larval Coquillettidia perturbans in south-central Michigan.

Seasonal abundance of immature Coquillettidia perturbans was studied at 3 sites in south-central Michigan in 1987 and 1988. A modified boat bilge pump and funnel separators proved to be good sampling and sorting devices. Pupae were found in late May to early July, first and second instars were most abundant in July and August, while third and fourth instars were present year-round. Overall, larvae were most numerous in August and September, indicating that Cq. perturbans has a single generation per year in Michigan, with midsummer to fall as the main period of larval development, and with third and fourth larval instars as overwintering stages. Parasitism of Cq. perturbans larvae by a previously undiscovered mermithid nematode was documented at 2 sites in late summer.

A broad evaluation of B.t.i. for black fly (Diptera: Simuliidae) control in a Michigan river: efficacy, carry and nontarget effects on invertebrates and fish.

Efficacy for black fly control, carry and nontarget effects of B.t.i. (Teknar HP-D), applied in the Betsie River, Michigan, were studied in June 1988. Black fly mortality was high (approximately 100%) for a 2,200 m stretch downstream from the application site, declined to 30% at 3,200 m, and was nil at 4,500 m. Drift of black flies greatly increased after application at a downstream site, but did not change at an upstream site. There were no detectable nontarget effects of B.t.i. application on: 1) invertebrate macro- or micro-drift; 2) numbers of invertebrates in benthic Surber samples; 3) mortality or feeding of drifting and nondrifting insects; 4) growth or mortality of caged Stenomena sp. larvae; 5) invertebrate functional group composition; 6) mortality or weight change of caged rock bass; or 7) fish numbers, species composition, length-weight (rock bass only) relationships or rock bass diet. Sampling of Rheotanytarsus sp. midges on natural substrates indicated low (27%) mortality owing to B.t.i. at only 100 m downstream from the application site, with negligible mortality at all other downstream and upstream sites. This information, combined with no pronounced changes in numbers of midges in macro-drift after application, indicated that midge populations were not adversely affected by B.t.i. in the study.

The digestion of protein and carbohydrate by the stream detritivore, Tipula abdominalis (Diptera, Tipulidae).

The digestive system of larvae of Tipula abdominalis (Diptera, Tipulidae), a stream detritivore, is poorly adapted for the digestion of the major polysaccharides in its diet, but well adapted for the digestion of protein. These crane fly larvae are unable to digest the major cell wall polysaccharides of higher plants, i.e., cellulose, hemicellulose and pectin. The only polysaccharides toward which the midguts of T. abdominalis exhibited any activity were α-amylose and laminarin, indicating that polysaccharide digestion is restricted to α-1,4-and ß-1,3-glucans. The most concentrated source of these two classes of carbohydrates in submerged leaf litter would be associated fungal tissue. The midgut of T. abdominalis is strongly alkaline throughout, with a maximum pH near 11.5 in a narrow zone near the midpoint. Proteolytic activity in the midgut is extraordinarily high, and the pH optimum for midgut proteolytic activity is above 11. We conclude that the high alkalinity and high proteolytic activity observed in T. abdominalis larvae are manifestations of a highly efficient protein-digesting system, a system of crucial importance to a nitrogen-limited organism which must derive its nitrogen from a resource in which much of the limited nitrogen present is in a "bound" form in complexes of proteins with lignins and polyphenols.