(Diptera: Calliphoridae) Oviposition

DIRECT INJURY, MYIASIS, FORENSICS

Environmental Factors Associated With Phormia regina (Diptera: Calliphoridae) Oviposition MAUREEN C. BERG

AND

M. ERIC BENBOW1

Department of Biology, University of Dayton, 300 College Park Drive, Dayton, OH 45469 Ð2320

J. Med. Entomol. 50(2): 451Ð457 (2013); DOI: http://dx.doi.org/10.1603/ME12188

ABSTRACT The period of insect activity (PIA) contributes information to estimates of the minimum postmortem interval in forensic investigations and begins with blow ßy attraction and oviposition on a resource such as carrion or a human corpse. Incorrectly estimating nocturnal oviposition could alter PIA estimates by up to at least 12 h; however, the extent of this difference in PIA would depend on environmental and geographic factors. To date, the literature on the extent and frequency of blow ßy nocturnal oviposition is conßicting. Our objectives were as follows: 1) to measure the effects of artiÞcial lighting and beef liver bait height above ground on nocturnal and diurnal oviposition, and 2) to monitor oviposition through the night on swine carcasses exposed to the environment at dusk in different habitats over 3 yr. We documented no consistent nocturnal oviposition in any trial using beef liver or on carcasses in different habitats and seasons. There were statistically signiÞcant effects of light and height of bait above the ground on diurnal oviposition of Phormia regina (Meigen) in August of 2009, the only month with mean night temperatures ⬎20⬚C. In August there also was signiÞcantly greater diurnal oviposition on liver bait placed on the ground compared with bait elevated 1 m. Our results suggest that nocturnal oviposition is rare in the natural environment. However, we found enhanced diurnal oviposition of P. regina under conditions of ambient temperatures ⬎20⬚C the night before oviposition. Additional studies are needed to better understand the ecological mechanisms governing blow ßy oviposition important to forensic entomology. KEY WORDS Calliphoridae, postmortem interval, period of insect activity, decomposition ecology, forensic entomology

In criminal cases, the postmortem interval (PMI) is the time between death and body discovery. Minimum PMI (PMImin) estimates are often closely aligned to the period of insect activity (PIA) in investigations where arthropods are used as evidence (Amendt et al. 2007). The PIA begins with blow ßy (Diptera: Calliphoridae) attraction to a resource that often results in oviposition or larviposition that can occur within minutes, hours, or days after death (Amendt et al. 2007, Tomberlin et al. 2011b); however, this event can be delayed in various ways, such as concealment or storage of the remains (Tomberlin et al. 2011a). Thus, the timing of oviposition is a key factor of the PIA that assists in estimates of entomologically-based PMImin (Byrd and Castner 2010). In forensic cases involving arthropod evidence, it is often assumed that blow ßies do not oviposit at night, even though published studies suggest substantial variability in this event under natural settings (Table 1). From a review of the literature, nocturnal oviposition has been reported to not occur, or to be highly reduced, under darkened conditions both in the Þeld and under laboratory conditions (Table 1). It also has 1

Corresponding author, e-mail: [email protected].

been suggested that variability in nocturnal oviposition is related to the degree of artiÞcial lighting at any speciÞc study site or under a range of experimental conditions (Table 1). An incorrect assumption of nocturnal oviposition, or an under appreciation of its variability, could potentially change an estimated PIA by up to at least 12 h depending on environmental conditions and geographic location (Greenberg 1990). Studies of blow ßy nocturnal oviposition have been conducted in several geographic regions and habitat types (Table 1). Amendt et al. (2007) reported nocturnal oviposition ⬇33% of the time in a laboratory setting, with no oviposition documented using hedgehog carcasses in the Þeld at night. Similarly, another study reported nocturnal oviposition on swine carcasses in only 33% of Þeld trials, and also documented in a series of lab studies, that Lucilia sericata (Meigen) (Diptera: Calliphoridae) was not capable of ßight in total darkness (Zurawski et al. 2009). Faucherre et al. (1999) reported oviposition in a completely dark cave (10 m deep) on both a human body and pork meat bait. Baldridge et al. (2006) measured blow ßy nocturnal oviposition by comparing bait types (i.e., white rats, ground sirloin beef, and Yorkshire cross pigs) and habitats (i.e., urban and rural). The urban setting was

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Table 1. A summary of previous studies of blow fly nocturnal oviposition including bait type, artificial light, exposure time, and results (i.e., conditions in which oviposition either did or did not occur) Citation

Location

Bait

Light source

Greenberg (1990)

Illinois, USA Rat, ground Sodium vapor beef street lamps

Tessmer et al. (1995)

Louisiana, USA

Faucherre et al. Switzerland (1999) Singh and India Bharti (2001)

Exposure 0100Ð0400 hours, 2100Ð0000 hours

Chicken

Various artiÞcial 1300Ð2000 hours, lights 2100Ð0500 hours, 0600Ð1300 hours

Pork meat

Natural

Results 33% trials with nocturnal oviposition No nocturnal oviposition

12 d in a dark cave

Oviposition after 12 d Mutton Natural 2200Ð0300 hours 33% trials with nocturnal oviposition Williams (2002) South Africa Rat, pork Natural 2 h in the dark Oviposition occurred meat 31Ð35% lab/13% Þeld Baldridge et al. Texas, USA Rat, ground Natural 1800, 2100, 0000, and One oviposition (2006) beef, pig 0600 hours event in 200 h of bait presentation Amendt et al. Germany Hedgehog Natural 2230 until 0430 hours Two oviposition (2007) (Þeld), 2100Ð0700 events in the hours (lab) laboratory, zero in the Þeld Zurawski et al. Michigan, Swine Natural halogen 2 h before sunset for 33% trials with (2009) USA 1 wk nocturnal oviposition Kirkpatrick and Illinois, USA Ground Halogen lights 1200Ð0300 hours for Oviposition events Olson (2007) Beef 6 night under artiÞcial lighting with temperature ⬎26⬚C Stamper and Ohio, USA Rat Street lamps 1 h after sunset-1 h No nocturnal Derby (2007) after sunrise oviposition

Species studied L. sericata, C. vicina, P. regina C. macellaria, Lucilia (Phaenicia) sericata, Sarcophaga bullata (Parker) C. vicina C. rufifacies, Chrysomya megacephala (F.), C. vicina L. sericata, C. putoria (Wiedemann), C. chloropyga (Wiedemann) Cochliomyia macellaria (F.), L. coeruleiviridis (Macquart) L. sericata

L. sericata, C. macellaria, C. vicina L. eximia (Wiedemann), C. macellaria

Not reported

This table was modiÞed and expanded from Zurawski et al. (2009) and Stamper and Derby (2007).

characterized by substantial artiÞcial light. Fourteen rat carcasses were placed at the urban site and attracted adult blow ßy activity but without oviposition activity, and no ßies were observed on the baits between 2200 and 0600 hours; adults were observed ßying around and sometimes landing on the carcass (Baldridge et al. 2006). Kirkpatrick and Olson (2007) tested halogen light effects on this behavior and found that nocturnal oviposition occurred under artiÞcial light at temperatures ⬎26⬚C. Ground beef bait not exposed to artiÞcial lighting did not have nocturnal oviposition by ßies. Greenberg (1990) placed rat carcasses and ground beef bait near sodium vapor lamps in a residential suburb. He reported small numbers of eggs at sunset in 33Ð36% of trials when ground beef bait was exposed between midnight and 0400 hours during summer months; however, there was no oviposition on carcasses from 2100 hours to midnight. In addition, Stamper and Derby (2007) reported no nocturnal oviposition on 48 rat carcasses exposed at night in both unlit and artiÞcially lighted areas of Cincinnati, OH. A summary of these and additional studies related to nocturnal oviposition are given in Table 1. There is substantial variability in the results of studies that have measured blow ßy nocturnal oviposition. This variation in study Þndings may be attributed to the differences in bait types (e.g., whole carcasses versus butchered meat), incomplete understanding of the type or extent of artiÞcial lighting in Þeld settings,

and recognized limitations of unnatural conditions inherent to laboratory experiments. Further, little empirical data are available on the environmental factors that may inßuence the timing and extent of diurnal oviposition when a carcass is exposed to the environment at sunset on the preceding evening. This is important for understanding how variable both nocturnal and diurnal oviposition can be under naturally heterogenous habitat and climate conditions, and could provide more basic biological understanding that could be used to improve the application of this information within the forensic sciences (Tomberlin et al. 2011a,b). In this paper, we provide a series of Þeld studies that experimentally addressed the importance of artiÞcial lighting and bait height above ground on nocturnal and diurnal oviposition. Secondly, we measured the environmental variables that may inßuence diurnal oviposition the day after carcasses were exposed the preceding evening. Materials and Methods Study Sites. The artiÞcial light and height experiments, and some carcass measurements (see Nocturnal Oviposition on Carcasses), were conducted at Morris Bean Reserve (39⬚ 45⬘53.67⬙ N, 83⬚ 54⬘41.12⬙ W) of Greene County, OH as part of a larger project measuring the decomposition process of vertebrate carcasses. The study site is described in detail by Lewis

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BERG AND BENBOW: OVIPOSITION OF P. regina

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Fig. 1. Oviposition bait stands (with plastic boxes on top) used in the light and height-above-ground experiment in the forested habitat of the Morris Bean Reserve, Greene County, OH. The stands were 1 m high above the ground.

and Benbow (2011). Brießy, the forested lot was 12.2 ha surrounded by agricultural Þelds and with a small tributary stream running along the reserve that empties into the Little Beavercreek River. The predominant trees were honey locust (Gleditsia triacanthos L.) and several maple species (Acer spp.). The most common subcanopy cover was Amur honeysuckle [Lonicera maackii (Rupr.) Maxim.]. Additional blow ßy carcass oviposition measurements of swine carcasses in 2010 and 2011 took place in a private forested lot (dominated by Acer spp. and Quercus spp.) with a small Þrst order stream running through the habitat, located near Bellbrook, OH and ⬇12 km away from the Morris Bean site. Artificial Light and Bait Height Effects on Oviposition. Three plots, ⬎ 30 m apart within an area of ⬇120 m2, inside the Morris Bean reserve were selected for light treatment conditions by using six 1-m-high bait stands per plot. Each stand was a 1-m wood post with a washed, 1-liter clear plastic box (no. 1642; Sterilite Corporation, Townsend, MA) of 4.6 cm in length by 21 cm in width by 12.4 cm in height, attached to the top by using screws (Fig. 1). Thirty-three holes (3 cm in diameter) were drilled into each plastic box that also contained a Styrofoam bowl with 35.0 ⫾ 2.0 g of beef liver. Beef liver was purchased fresh from a local grocery store during each month of study and the same store was used for all purchases. For each light experiment, the bait was not replaced throughout the

night, but fresh liver was added to each bowl 2 h before sunrise. Light experiments were conducted in July, August, September, October of 2009, and June of 2010. Beef liver bait (n ⫽ 3/treatment) was placed in the plastic boxes under three light treatments: high (six lux), low (three lux), and control (0 lux). For each treatment, one 8D Family Sized LED Lantern (model no. 4345Ð701; Coleman, Wichita, KS) was strung by rope 1 m off of the ground. To measure the effect of bait elevation on oviposition, we monitored oviposition on bait elevated at 1 m to compare with oviposition activity associated with bait in the same type of boxes on the ground. Three ground boxes and three 1-m stands were staked into the ground so that all boxes made a circle around the hanging lantern. The location of each box randomly was selected to determine the amount of artiÞcial light exposed to the liver bait based on distance and angle from the baits. The distances for each light treatment were as follows: high (1.3 m), low (3.7 m), and control (no light source and either 1.3 or 3.7 m to match the light treatments). Differences in light intensity between the elevated bait and that on the ground were negligible, so that the elevated and ground baits received the same amount of light within a treatment. Beef liver was placed in the boxes 2 h before sunset each month within 3Ð 4 d of a new moon. Sunset and sunrise were deÞned using data from the National Oceanic and Atmosphere Administration (NOAA).

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Carcasses were checked hourly from 2 h before sunset until 2 h after sunset and then again beginning 2 h before sunrise until 2 h after sunrise, similar to methods describe by Zurawski et al. (2009). At 2 h before sunrise an additional set of fresh liver was placed in the boxes. This bait then was monitored for another 4 h until 2 h after sunrise. Finally, 24 h after initial exposure, the bait was observed again for blow ßy oviposition. Nocturnal Oviposition on Carcasses. At the Morris Bean site another study was conducted to only measure nocturnal oviposition on Sus scrofa (swine) carcasses weighing 16 ⫾ 2 kg. The carcasses were purchased from a local farm immediately after being euthanized by blunt force trauma to the head and immediately were double bagged in 1-mm-thick garbage bags that were sealed with duct tape to prevent any invertebrate access. The carcasses were from animals where we did not know the dose regimen of antibiotics, but they presumably were treated with some antibiotics during growth. The carcasses were transported and held at ambient temperature for 1Ð3 h before being placed at the study location. Each carcass was placed on a single 1-m2 plot randomly along one of six 50-m transects running north to south. There were a total of 30 plots 45 ⫾ 35 m apart (Lewis and Benbow 2011). Carcasses were exposed at 2 h before sunset deÞned by NOAA on 15 April 2009 (1,814), 22 July 2009 (1,858), 11 November 2009 (1,623) and 8 February 2010 (1,704), for spring (n ⫽ 6 carcasses), summer (n ⫽ 6), autumn (n ⫽ 3), and winter (n ⫽ 3) experiments, respectively. Each carcass was placed under a wood exclosure cage (0.6 by 0.95 by 0.6 m) with 2.5-cm mesh chicken wire to prevent disturbance by vertebrate scavengers (e.g., raccoons, coyotes, vultures). These carcasses were monitored on the same schedule as the artiÞcial light study described above. At a separate site near Bellbrook, OH, four and three swine carcasses were used to measure nocturnal oviposition on 28 July 2010 and 26 July 2011, respectively. These carcasses were purchased from the same farm as described above. In 2010, two of the carcasses were placed in a terrestrial habitat, whereas the other two were placed on the bank of a small (⬇1 m wide) stream running through the forested lot. In 2011, three carcasses were placed in a terrestrial habitat ⬇12-m elevation above and 60 m south of the stream. The carcasses were monitored in the same time schedule described above. For both studies, any eggs present at each observation were collected and transported to the laboratory at the University of Dayton, Dayton OH, ⬇30 min from the study site. During each observation time, a ßashlight was used to make thorough and careful visual assessments of blow ßy eggs. For liver baits, the liver was moved to look around and on the sides for eggs. For the carcasses, all external surfaces were measured carefully, including gently lifting the body up to observe on the sides and underside of the carcass. We also would slowly open the mouth and probe the buccal cavity for eggs and thoroughly inspect behind the ears and in all crevices and folds in the swine skin.

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Eggs were counted individually under a stereoscope and individual masses were weighed to the 0.01 g to develop a regression model to predict egg number from egg mass (see below). All eggs then were reared for identiÞcation by using methods described in Benbow et al. (2013). Reared adult ßies were identiÞed to the lowest taxonomic level possible using a stereomicroscope (Whitworth 2006). Temperature was recorded every 15 min through the duration of the studies by using NexSens DS1921G micro-T data loggers (Fondriest Environmental Inc., Beavercreek, OH). Light, as lux, was measured using an Extech Lux Light Meter (Extech Instruments Corporation, Nashua, NH). Data Analysis. A linear regression model was developed for Phormia regina (Meigen) (Diptera: Calliphoridae) to predict egg number from egg mass. We used a one-way analysis of variance (ANOVA) to test for temperature differences among months and SpearmanÕs Rank Correlation to measure associations between temperature and oviposition egg density on liver bait. Lastly, we used a two-way repeated measures ANOVA with Bonferroni posttests as described by Motulsky (2003) to test for light, bait height above ground, and interaction effects on diurnal oviposition over the months of study. All analyses were done using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA). Power analyses were run using G*Power three (Faul et al. 2007). Results Artificial Light and Bait Height Effects on Oviposition. From all treatments and replicates only 90 eggs were collected from beef liver bait. Eggs were collected within 2 h before sunset with no oviposition documented during the night hours (deÞned here as 2 h after sunset to 2 h before sunrise, times deÞned by NOAA). All eggs were reared and identiÞed as P. regina. There was a strong and signiÞcant relationship between egg number and egg mass (y ⫽ 1,776x ⫺ 10.53; R2 ⫽ 0.98; P ⬍ 0.0001; F ⫽ 1,722; n ⫽ 45 egg masses), allowing us to use egg mass to extrapolate egg density for each diurnal oviposition event. August was signiÞcantly warmer during both the day and night except compared with day temperatures of June of 2010 (Fig. 2): Day, F ⫽ 269; df ⫽ 4,15; P ⬍ 0.0001 (Bonferroni posttests P ⬍ 0.05 except August 2009 versus June 2010); night, F ⫽ 203; df ⫽ 4,15; P ⬍ 0.0001 (all Bonferroni posttests at P ⬍ 0.05). There was a nearly signiÞcant positive correlation between mean night temperature and the subsequent day diurnal oviposition egg density (Spearman r ⫽ 0.90, P ⫽ 0.08), but no signiÞcant relationship between diurnal oviposition and the preceding mean day temperature (Spearman r ⫽ 0.82, P ⫽ 0.13). We found no signiÞcant effect of artiÞcial light on diurnal egg density after accounting for the signiÞcant effects of month and height (Table 2). Interestingly, there was signiÞcantly greater diurnal oviposition activity with bait on the ground compared with bait at 1 m elevated only under high light conditions (Bonferroni posttest P ⬍ 0.05). The overall month effect


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Fig. 2. Mean (SE) ambient temperature conditions at night and the next day for the light and height-above-ground oviposition experiment. Numbers above the columns represent the mean egg density (number of eggs per gram liver) collected from each diurnal oviposition event the next day after exposure. Both day and night temperature were signiÞcantly warmer in August compared all months, except for day temperatures of June of 2010 that were statistically the same as August of 2009 (see text for statistics).

was driven by signiÞcantly higher oviposition activity during August of 2009 under high light conditions with bait on the ground (Bonferroni posttests P ⬍ 0.05) (Fig. 3). In August, this resulted in a 54% difference between high light, and low light and control, and a 175% increase for bait on the ground compared with 1 m above ground. There was less diurnal oviposition during the other months when mean nocturnal temperatures ranged from 10 to 20⬚C. However, when mean night temperatures were ⬍10⬚C there was no oviposition the next day. Nocturnal Oviposition on Carcasses. During the July 2009 trial, there was no blow ßy activity on any carcass until 4 h after initial exposure (2 h after sunset) even though it was raining and cold (15⬚C). Under these weather conditions, we observed one unidentiÞed female ovipositing in the snout of a single carcass. The eggs were reared and identiÞed as P. regina. This was the only nocturnal ßy activity observed for the rest of that night or any of the other trials in 2010 and 2011 from either habitat. For the remaining trails there was no rain and all insect activity ceased approximately 3 hr after exposure (1 h after sunset), and there was no documented nocturnal oviposition. Blow ßies returned to the carcasses (along with other insects) no sooner than 2 h after sunrise. Discussion In this study, carcasses in conditions of average night temperatures ⬎20⬚C had greater subsequent day Table 2. Two-way repeated measures ANOVA statistics that tested for the effects of bait height above ground and light treatments on diurnal egg density (mean number of eggs per gram liver) over the months of study (as the repeated measure) Effect

Sum of squares

df

F-value

P value

Light Height Light ⫻ height Month Error Total

57,446 124,701 19,721 292,230 65,987 560,086

2 1 2 9 9 23

0.89 17.01 1.35 4.43

0.4459 0.0026 0.3083 0.0186

The power for this analysis was 0.436.

diurnal oviposition than other months. This elevated temperature is consistent with Þndings by Kirkpatrick and Olson (2007), who only observed nocturnal oviposition at temperatures ⬎26⬚C. This may suggest a potential hypothesis of a temperature threshold for nocturnal oviposition and elevated diurnal oviposition the next day; however, this requires additional investigation. Similarly, Zurawski et al. (2009) reported a positive multiple regression linear relationship of next-day diurnal oviposition with wind speed, temperature, and humidity, suggesting that other abiotic factors are important to diurnal oviposition. In our study, July and September 2009 mean night temperatures were ⬍20⬚C but carcasses in those months were found with lower blow ßy oviposition the next day (compared with August of 2009). During this trial, there was a short, intense rain event (⬇2Ð 4 cm/h) during the day that could have affected oviposition. Rainfall has been shown to inhibit adult blow ßy activity and even prolong larval development (Mahat et al. 2009). Mahat et al. (2009) reported that freshly hatched larvae of Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) always were found 1Ð2 d later than expected after rainfall. Although nocturnal oviposition was not observed in this study, bait that was exposed to higher artiÞcial light intensities were found with more blow ßy egg densities the next day when compared with bait under reduced artiÞcial and ambient light conditions; however, this was only signiÞcant during the warmest month. This could be the result of females being attracted to the carcass during the night, and by sunrise they are fully gravid and can ßy to the carcass, allowing oviposition to occur after daybreak. They may also have been nutrient deprived with associated enhanced sensitivity to resource odors. It is known that both reproductive condition and nutritional status inßuences the responsiveness and behavior of other blow ßy species, including the blow ßies P. regina and Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae) (Browne 1993) and L. sericata (Tomberlin et al. 2013). Further, Ashworth and Wall (1995) reported that a large number of L. sericata and P. regina adults, without an acquired protein source, were more likely

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Fig. 3. Effects of (A) height above ground (1 m above versus on the ground) and (B) artiÞcial light levels on next day diurnal oviposition as mean (SE) egg density for each month. The asterisks indicate signiÞcant Bonferroni posttest (P ⬍ 0.05) differences for high light and bait on the ground compared with the other treatments only in August. There were no other signiÞcant posttest differences between treatments for other months. See Table 2 for main effects and interaction statistics.

to come in contact with protein when it was associated with an odor pulse cue. Therefore, it is possible that blow ßies most attracted to the bait were fully gravid, and used odor cues to locate the bait until there was enough light for ßight and oviposition. Additional studies on this interaction between physiology, behavior, and oviposition are warranted. During the swine oviposition trial in July of 2009, there was heavy rain (⬇ 2Ð 4 cm/h) during the night (deÞned here as ⬎2 h post-NOAA sunset). There was only one observed event of a single blow ßy on a swine carcass 2 h after sunset. The female adult hopped and crawled away from the carcass when it was disturbed, but did not take ßight. This observation, out of several monthly trials in different seasons by using swine carcasses, is similar to previous research (Baldridge et al. 2006, Zurawski et al. 2009). This observation occurred 4 h after the carcasses were placed into the plot at 2200 hours. The heavy rain may have discouraged any additional blow ßy activity on the carcasses. Zurawski et al. (2009) reported that L. sericata adult males and females were not capable of ßight in the lab under total darkness. They noted that when female adults were launched from investigator eye level height under total darkness, there was no audible wing beating sound after the launch (Zurawski et al. 2009). However, Faucherre et al. (1999) reported that Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) eggs were found on a human body 10 m inside of a completely dark cave with ambient temperature conditions of 5⬚C. They conÞrmed this by placing fresh pork meat and liver inside the cave at the same location and found eggs on the bait 12 d later; however, this oviposition activity could have also been from ßies walking or crawling to the resource.

We did not observe any nocturnal oviposition in 2010 by using swine carcasses in terrestrial and adjacent to aquatic environments. Blow ßies colonized carcasses within the Þrst hour of exposure, but all insect activity terminated no later than 1 h after sunset. Insects returned to the carcass no later than 2 h after sunrise. This pattern of blow ßies leaving the carcass soon after sunset, and returning to the carcass after sunrise, was reported in several studies (Tessmer et al. 1995, Baldridge et al. 2006, Stamper and Derby 2007). Similar to Stamper and Derby (2007), we did not observe nocturnal oviposition between the two habitats, under the presence and absence of artiÞcial light, adjacent to a stream, and using both beef liver and swine carcasses as bait, suggesting that nocturnal oviposition activity is very rare under several environmental conditions and among months and years. We conÞrm reports that oviposition does occur immediately before and during sunset but does not begin again until several hours after sunrise during this study in southwestern Ohio. This further supports the assumption that blow ßies will rarely oviposit on a carcass at night, even with moderate levels of artiÞcial light. Although it requires additional study, we report that there is approximately a 17% chance (one out of six carcasses with evening oviposition in July of 2009) that early nightfall (2 h after sunset) oviposition can occur during rainfall under relatively mild temperatures during midsummer in southwest Ohio. Diurnal oviposition on carcasses was consistent with the Þndings of the liver experiment. In 2009, 2010, and 2011, oviposition occurred on carcasses no later than sunset and no earlier than 2 h after sunrise. This study demonstrates that early sunset and sunrise oviposition by blow ßies varies greatly even within the same habitat,


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and the variability because of location, temperature, and light should be taken into consideration when making estimates of the PIA in forensic investigations. We also encourage the use of replicate carcasses or bait in future entomological studies of carrion decomposition to provide the necessary information for strong meta-analyses as advocated in two recent papers (Michaud et al. 2012, Tomberlin et al. 2012). A more comprehensive understanding of blow ßy oviposition behavior can have important implications to the Þeld of forensic entomology. Acknowledgments We thank the Greene County Park District, Xenia, OH for access and permission to conduct this study on its property, and the University of Dayton Institutional Animal Care and Use Committee for evaluating our study procedures and determining that purchasing carcasses without any intervention or guidance did not require approval by the committee. The University of Dayton Research Council, Graduate School and Department of Biology provided funding for these studies. We also thank A. Lewis, T. Blair, M. Shoda, J. White, M. Diaz, A. Lai, J. Lang, A. Gansel, and C. Teter for Þeld assistance, and the Blair family for permission to conduct the second study on their property. We are grateful to J. Pechal for reviewing an earlier draft of this paper.

References Cited Amendt, J., R. Zehner, and F. Reckel. 2007. The nocturnal oviposition behavior of blowßies (Diptera: Calliphoridae) in Central Europe and its forensic implications. Forensic Sci. Int. 175: 61Ð64. Ashworth, J. R., and R. Wall. 1995. Effects of ovarian development and protein deprivation on the activity and locomotor responses of the blowßy, Lucilia sericata, to liver odour. Physiol. Entomol. 20: 281Ð285. Baldridge, R. S., S. G. Wallace, and R. Kirkpatrick. 2006. Investigation of nocturnal oviposition by necrophilous ßies in Central Texas. J. Forensic Sci. 51: 125Ð126. Benbow, M. E., A. J. Lewis, J. K. Tomberlin, and J. L. Pechal. 2013. Seasonal necrophagous insect community assembly during vertebrate carrion decomposition. J. Med. Entomol. (in press). Browne, L. B. 1993. Physiologically induced changes in resource-oriented behavior. Annu. Rev. Entomol. 38: 1Ð25. Byrd, J., and J. Castner (eds.). 2010. Forensic entomology: the utility of arthropods in legal investigations. CRC, Boca Raton, FL. Faucherre, J., D. Cherix, and C. Wyss. 1999. Behavior of Calliphora vicina Diptera, Calliphoridae under extreme conditions. J. Insect Behav. 12: 687Ð690. Faul, F., E. Erdfelder, A.-G. Lang, and A. Buchner. 2007. G*Power 3: A ßexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav. Res. Methods 39: 175Ð191. Greenberg, B. 1990. Nocturnal oviposition behavior of blow ßies (Diptera: Calliphoridae). J. Med. Entomol. 27: 807Ð 810.

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Kirkpatrick, R. S., and J. K. Olson. 2007. Nocturnal light and temperature inßuences on necrophagous, carrion-associating blow ßy species (Diptera: Calliphoridae) of forensic importance in Central Texas. Southwest. Entomol. 32: 31Ð36. Lewis, A. J., and M. E. Benbow. 2011. When entomological evidence crawls away: Phormia regina en masse larval dispersal. J. Med. Entomol. 48: 1112Ð1119. Mahat, N. A., Z. Zafarina, and P. T. Jayaprakash. 2009. Inßuence of rain and malathion on the oviposition and development of blowßies (Diptera: Calliphoridae) infesting rabbit carcasses in Kelantan, Malaysia. Forensic Sci. Int. 192: 19 Ð28. Michaud, J.-P., K. G. Schoenly, and G. Moreau. 2012. Sampling ßies or sampling ßaws? Experimental design and inference strength in forensic entomology. J. Med. Entomol. 49: 1Ð10. Motulsky, H. J. 2003. Prism 4 Statistics Guide - Statistical analyses for laboratory and clinical researchers, GraphPad Software Inc., San Diego, CA. Singh, D., and M. Bharti. 2001. Further observations on the nocturnal oviposition behaviour of blow ßies (Diptera: Calliphoridae). Forensic Sci. Int. 120: 124 Ð126. Stamper, T., and R. W. Derby. 2007. The nocturnal oviposition behavior of carrion ßies in rural and urban environments: methodological problems and forensic implications. Can. Soc. Forensic Sci. J. 40: 173Ð182. Tessmer, J. W., C. L. Meek, and V. L. Wright. 1995. Circadian patterns of oviposition by necrophilous ßies (Diptera: Calliphoridae) in southern Louisiana. Southwest. Entomol. 20: 439 Ð 445. Tomberlin, J. K., M. E. Benbow, A. M. Tarone, and R. Mohr. 2011a. Basic research in evolution and ecology enhances forensics. Trends Ecol. Evol. 26: 53Ð55. Tomberlin, J. K., R. Mohr, M. E. Benbow, A. M. Tarone, and S. VanLaerhoven. 2011b. A roadmap for bridging basic and applied research in forensic entomology. Annu. Rev. Entomol. 56: 401Ð 421. Tomberlin, J., J. Byrd, J. Wallace, and M. Benbow. 2012. Assessment of decomposition studies indicates need for standardized and repeatable research methods in forensic entomology. J Forensic Res. 3: 147. Tomberlin, J. K., T. L. Crippen, A. M. Tarone, B. Singh, K. Adams, Y. H. Rezenom, M. E. Benbow, M. Flores, M. Longnecker, J. L. Pechal, et al. 2012. Interkingdom responses of ßies to bacteria mediated by ßy physiology and bacterial quorum sensing. Anim. Behav. 84: 1449 Ð1465. Whitworth, T. 2006. Keys to the genera and species of blow ßies (Diptera: Calliphoridae) of America north of Mexico. Proc. Entomol. Soc. Wash. 108: 689 Ð725. Williams, K. A. 2002. Spatial and temporal occurrence of forensically important South African blowßies (Diptera: Calliphoridae). MS thesis, Rhodes University, Grahamstown, South Africa. Zurawski, K. N., M. E. Benbow, J. R. Miller, and R. W. Merritt. 2009. Examination of nocturnal blow ßy (Diptera: Calliphoridae) oviposition on pig carcasses in MidMichigan. J. Med. Entomol. 46: 671Ð 679. Received 21 August 2012; accepted 22 December 2012.