Evaluating the impact of hot water killing larvae on gene expression using the transformer gene in Cochliomyia macellaria (Diptera: Calliphoridae).

In forensic entomology, determining the age of a larva from a body to estimate time since death is commonly performed through the measurement of a physical trait. Gene expression has been studied as an alternative age estimation approach, but the storage conditions required for these studies are different than those used in forensic entomological casework. Studies analyzing gene expression prioritize the preservation of RNA, which requires fresh tissue and ultra-cold storage. Casework, in contrast, utilizes hot water killing specimens that may not be analyzed for a long period after collection. In the current study, the impact of hot water killing on gene expression was assessed for larval samples of the forensically important blow fly, Cochliomyia macellaria. Successful amplification of the sex-determining gene, transformer, was tested across larvae ranging in size from 3.22 to 16.85 mm in length after storage times of 1-2 weeks, 4-5 weeks, and 8-9 weeks at 4°C in RNAlater. Larvae hot water killed were processed in tandem with larvae stored live to allow for a direct assessment of the impact of boiling on gene expression. As expected, the transformer gene was successfully amplified in all larvae stored live. For the hot water-killed larvae, the success rate was only slightly lower, with 3 out of 75 larvae not generating a sex-specific band pattern. The results show gene expression can be used for hot water-killed samples, though future work across different genes, species, and extending to quantitative gene expression methods is needed.

Initial Work on Sex-Specific Growth Curve Guidelines for Forensically Important Blow Flies Using Chrysomya megacephala (Diptera: Calliphoridae) Entire Age Cohorts.

Sex is a factor influencing development in many insect species, but is not widely studied in forensically important blow flies. If sex influences blow fly development, knowing the sex of a larva from a corpse can increase precision in estimates of that larva's age. The improved prediction of larval age will make estimates of time since death using entomological evidence better. Larvae lack sexually dimorphic morphological characteristics, so sex is not immediately known visually. To generate sexually dimorphic reference growth curves, a subsample must be large enough to ensure enough males and females are present for comparison. Using two entire age Chrysomya megacephala (Fabricius) cohorts, we evaluated the minimum sample number needed to have enough individuals of both sexes for comparison using 95% prediction intervals. Through a simulation of three trials of 1000 random replicates, we determined that a sample size of 19 would prevent any instance of a comparison not occurring because of insufficient sampling from one sex. As the current method for molecular sex determination can be expensive, we also compared how the results of various subsampling percentages compare those of the entire age cohorts. We found that subsampling at least 50% of an entire cohort leads to almost identical results in comparison to the entire age cohort. Together, these findings will help establish guidelines for generating sex-specific reference growth curves. A uniform approach to generating these sex-specific growth curves will lead to more consistency in age estimates made from them.

To play or not to play? The relationship between active video game play and electrophysiological indices of food-related inhibitory control in adolescents.

Sedentary behaviors, such as computer use and sedentary video games, are barriers to physical activity, contribute to overweight and obesity among adolescents, and can adversely affect eating behaviors. Active video games may increase daily physical activity levels among adolescents and improve food-related inhibitory control. We compared the effects of acute bouts of active and sedentary video gaming on event-related potential (ERP) indices of food-related inhibitory control, energy expenditure, and ad libitum eating. In a within-subjects design, 59 adolescent participants (49% female, Mage  = 13.29 ± 1.15) completed two separate counterbalanced, 60-min long video gaming sessions separated by seven days. Immediately after, participants completed two go/no-go tasks with high- and low-calorie images and N2 and P3 ERP amplitudes were measured. Participants also completed a Stroop task and were given high- and low-calorie snacks to consume ad libitum. Results indicated that active relative to sedentary video games significantly increased energy expenditure on multiple measures (e.g., METs, heart rate, kcals burned) and participants consumed more calories after the active compared to the sedentary video game session. N2 amplitudes were larger when participants inhibited to high- compared to low-calorie foods, suggesting that high-calorie foods necessitate increased the recruitment of inhibitory control resources; however, there were non-significant differences for the N2 or P3 amplitudes, accuracy or response times, and Stroop performance between active versus sedentary video game sessions. Overall, sixty minutes of active video gaming increased energy expenditure and food consumption but did not significantly alter neural or behavioral measures of inhibitory control to food stimuli.

First Use of an Entire Age Cohort to Evaluate the Role of Sex in the Development of the Forensically Important Chrysomya megacephala (Diptera: Calliphoridae).

The age of a blow fly larval specimen found on a body can be used to estimate a minimum postmortem interval by comparing a physical feature of a larva (e.g., length) to a reference growth curve created under similar conditions. A better understanding of factors known to influence growth rates would lead to more precise estimates of larval age. A factor known to influence insect development, but almost always lacking in blow fly larval growth curves, is sex. We wanted to understand how sex might affect larval growth rates and therefore lead to more precise age estimates, in the forensically important blow fly Chrysomya megacephala (F.). We examined sex for an entire age cohort at 70 h and found on average, males were longer than females. To assess whether this difference would cause an investigator to interpret the results differently if comparing a larval specimen of one sex based on reference data from the other sex, we made sex-specific 95% prediction intervals. We did not find a difference when comparing a male specimen with the female prediction interval, but did find more females outside the interval than expected using the male interval. All females outside the male interval were shorter. Investigators commonly remove the largest individuals from a body to estimate age, so these females are less likely to be chosen. These results do not support the generation of sex-specific growth curves for this species, though this study's narrow scope means more information is required before making a final conclusion.

Small fruit flies sacrifice temporal acuity to maintain contrast sensitivity.

Holometabolous insects, like fruit flies, grow primarily during larval development. Scarce larval feeding is common in nature and generates smaller adults. Despite the importance of vision to flies, eye size scales proportionately with body size, and smaller eyes confer poorer vision due to smaller optics. Variable larval feeding, therefore, causes within-species differences in visual processing, which have gone largely unnoticed due to ad libitum feeding in the lab that results in generally large adults. Do smaller eyes have smaller ommatidial lenses, reducing sensitivity, or broader inter-ommatidial angles, reducing acuity? And to what extent might neural processes adapt to these optical challenges with temporal and spatial summation? To understand this in the fruit fly, we generated a distribution of body lengths (1.67-2.34 mm; n = 24) and eye lengths (0.33-0.44 mm; n = 24), resembling the distribution of wild-caught flies, by removing larvae from food during their third instar. We find smaller eyes (0.19 vs.0.07 mm2) have substantially fewer (978 vs. 540, n = 45) and smaller ommatidia (222 vs. 121 µm2;n = 45) separated by slightly wider inter-ommatidial angles (4.5 vs.5.5°; n = 34). This corresponds to a greater loss in contrast sensitivity (<50%) than spatial acuity (<20%). Using a flight arena and psychophysics paradigm, we find that smaller flies lose little spatial acuity (0.126 vs. 0.118CPD; n = 45), and recover contrast sensitivity (2.22 for both; n = 65) by sacrificing temporal acuity (26.3 vs. 10.8Hz; n = 112) at the neural level. Therefore, smaller flies sacrifice contrast sensitivity to maintain spatial acuity optically, but recover contrast sensitivity, almost completely, by sacrificing temporal acuity neurally.

The forensically important blow fly, Chrysomya megacephala (Diptera: Calliphoridae), is more likely to walk than fly to carrion at low light levels.

One factor that influences estimates of time since death using entomological evidence is whether or not blow flies nocturnally oviposit. Field studies focusing on egg laying have found it occurs on an inconsistent basis. A key but poorly understood factor in nocturnal oviposition is a blow fly's ability to locate carrion under low light levels. It has been speculated that blow flies are more likely to walk than fly to carrion during the night, but this has not been empirically tested. We directly compared guided walking versus flying using infrared sensors under low light levels in laboratory conditions for Chrysomya megacephala (F.) (Diptera: Calliphoridae), a blow fly previously described to be nocturnal. We found C. megacephala is more likely to walk than fly toward carrion under low light levels (p=0.016). We did not, however, find differences between males and females for walking (p=0.48) or flying (p=0.42) despite male C. megacephala possessing eyes better suited for increased light capture. These results demonstrate the need to better understand where blow flies go at night, as bodies found within a fly's walking distance are more likely to be colonized.

First report of Blaesoxipha plinthopyga (Diptera: Sarcophagidae) from a human corpse in the U.S.A. and a new state geographic record based on specimen genotype.

Carrion flies in the taxonomic family Sarcophagidae are often recovered from a human corpse. However, because such specimens are difficult to identify, the forensic literature on this taxon is quite limited compared with that of the commonly employed Calliphoridae. Faced with a sarcophagid larva that could not be identified microscopically from a death investigation in the state of Idaho, we generated cytochrome oxidase one DNA sequence data from the specimen. Comparison to a reference data set of forensically significant sarcophagids from Canada and the U.S.A. confirmed that this was the first discovery of Blaesoxipha plinthopyga in a human corpse in the U.S.A. and the first record of this species in Idaho. Because B. plinthopyga occurs from the Northern U.S.A. to the Neotropics, it is potentially useful for estimating time since death at many locations.