Identification of Oviposition Attractants of the Secondary Screwworm, macellaria (F.) Released from Rotten Chicken Liver

Junwei J. Zhu, Muhammad F. Chaudhury, Khanobporn Tangtrakulwanich & Steven R. Skoda

Journal of Chemical Ecology

ISSN 0098-0331 Volume 39 Combined 11-12

J Chem Ecol (2013) 39:1407-1414 DOI 10.1007/s10886-013-0359-z

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1 23 Author's personal copy

J Chem Ecol (2013) 39:1407–1414 DOI 10.1007/s10886-013-0359-z

Identification of Oviposition Attractants of the Secondary Screwworm, Cochliomyia macellaria (F.) Released from Rotten Chicken Liver

Junwei J. Zhu & Muhammad F. Chaudhury & Khanobporn Tangtrakulwanich & Steven R. Skoda

Received: 5 September 2013 /Revised: 25 September 2013 /Accepted: 13 October 2013 /Published online: 31 October 2013 # Springer Science+Business Media New York (outside the USA) 2013

Abstract The secondary screwworm, Cochliomyia macellaria elicited significant EAG responses from antennae of gravid (F.), is an important blowfly species affecting both livestock females. Oviposition assays showed that the 7-component and humans. It can transmit pathogenic disease agents blend stimulated gravid females to lay significantly more mechanically and is an agent of facultative , which than the other combinations tested. This 7-component blend leads to economic losses. The adult are attracted to may have potential for use in monitoring and sampling decomposing carcasses, carrion, or rotten meat in order to populations of secondary screwworm and their associated deposit their eggs, and the hatched larvae develop on these disease epidemiology. decaying organic materials. This research was aimed to identify volatiles emitted from rotten chicken livers that were Keywords Oviposition attractant . Blow flies . Forensic reported previously to attract gravid females. In laboratory veterinary pest . Solid phase microextraction . Livestock oviposition assays, gravid females laid significantly more disease . Economic entomology eggs on rotten livers than on fresh livers, and rotten chicken liver was more attractive than rotten beef liver. Volatiles from the two livers were collected using solid phase Introduction microextraction. Significantly different volatile profiles were detected from the rotten livers of beef and chicken. The secondary screwworm (SSW), Cochliomyia macellaria Electroantennography (EAG) was performed to determine (F.), belongs to the family . The species can be antennal responses to chemicals released from the most found in almost every known terrestrial throughout attractive chicken liver that are candidate oviposition the Western Hemisphere. In the United States, secondary attractants. Seven compounds from rotten chicken liver screwworms are of particular importance to both humans and the livestock industry because of large economic losses resulting from pathogen transmission and myiasis (Anderson J. J. Zhu (*) and Huitson 2004). Adults of secondary screwworm appeared USDA-ARS, Agroecosystem Management Research Unit, to be morphologically indistinguishable from the primary 305B Entomology Hall, UNL-EC, Lincoln, NE 68583, USA screwworm (PSW), (Coquerel) in e-mail: [email protected] earlier studies until 1933, when Laake and others discovered M. F. Chaudhury characteristic differences between these two species (Cushing USDA-ARS, Screwworm Research Unit, 105B Entomology Hall, and Patton, 1933; Laake et al. 1936). Their larval are UNL-EC, Lincoln, NE 68583, USA distinct in that PSW feeds and develops on living tissues of and humans (obligatory or primary myiasis) whereas K. Tangtrakulwanich Department of Entomology, University of Nebraska, Lincoln, SSW breeds in carrion or necrotic flesh (Laake et al. 1936). NE 68583, USA Although the larvae of SSW typically develop in decomposing carcasses, carrion, or rotten meat; they can also cause S. R. Skoda facultative myiasis by infesting wounds of animals and USDA-ARS, Screwworm Research Unit, Knipling-Bushland U. S. Livestock Research Laboratory, 2700 Fredericksburg Road, humans, often following invasion by primary myiasis- Kerrville, TX 78028, USA causing flies (Broce 1985; Mullen and Durden 2002). Recently Author's personal copy

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SSW has gained more recognition in the field of forensic refrigerated until used in the experiments. Livers were entomology as the principal species used to determine removed from the refrigerator and kept at room temperature postmortem interval estimations (Boatright and Tomberlin (25°C)andhumidity(55%R.H.)forabout1hr,and 2010). Furthermore, SSW has been considered as an invasive afterward were used as fresh liver; rotten livers were prepared pest that was intercepted from cargoes entering Asia from by leaving the liver pieces exposed at room temperature Mexico, Peru, and Panama (Deng et al. 2009). (25 °C) and humidity (55 % R.H.) for 3 days after purchase. In nature, adults of PSWand SSW occur together, but SSW greatly outnumbers PSW in liver, and in the traps baited with Landing Response to Oviposition Substrate Two sets of attractant that lures contain blends of compounds identified experiments were conducted using methods similar to those from infected wounds, “swormlures”, which are used described for the primary screwworm by Chaudhury et al. to monitor PSW adults (Coppedge et al. 1977; Jones et al. (2002) in wire mesh cages (30×30×30 cm) with solid metal 1976;Lindquist1938;Mackley1986). In the southern United base. In the first set, 25 eight-day-old male and female flies States, decaying animal remains (e.g., rotten beef and chicken were introduced in the cage using a 20 cm long and 1 cm livers) have been reported to be attractive to gravid females of diameter glass tube aspirator through the front opening that SSW, with -laying also observed (Byrd 1998). This was secured with a stretched cotton/polyester sleeve. Flies indicates that olfactory cues associated with these livers play were allowed to acclimatize in the cage for 10 min. a role in female SSW oviposition site selection. Meanwhile two clear plastic containers (11 cm top diam× The primary goal of this study was to isolate and identify 9 cm bottom diam×8 cm high) were prepared by lining the volatiles emitted from rotten beef and chicken livers that are bottom of each with a 9 cm filter paper on which 10 g of fresh attractive to gravid SSW females and stimulate oviposition. chicken liver were placed, and this served as the oviposition Results can contribute to the development of synthetic bait substrate. A piece of dental wick (1 cm) was placed on top of systems for monitoring this economically-important pest the liver in each container. The dental wick of one of the species for quarantine purposes and in control strategies to containers was treated with 1 ml of the 7-chemical blend reduce their abundance in urban recreational settings. (test) and that of the other was treated with an equal volume of ethyl alcohol (control). The prepared test and control containers then were introduced into the cage and placed on Methods and Materials the base of the cage diagonally and about 10 cm apart. behavior was observed for 15 min when the flies were Insects Secondary screwworms used in this study originated observed to land in the containers. Flies that landed in each from pupae obtained from a laboratory colony in the USDA- container were then captured by placing the container lid on ARS laboratory in Fargo, North Dakota, USA; progeny were top of the containers. Flies were immobilized by cooling in a maintained in a laboratory colony at the USDA-ARS, Lincoln, freezer, and the number trapped was recorded according to Nebraska,. Larvae were reared under laboratory conditions sexes for each container. (30 °C, 60 % RH and 16 L: 8 D) using an artificial diet The second set of experiments was conducted as in the first consisting of 60 g of spray-dried whole egg (California Spray series except 25 four-day-old females and 8-day-old females Dry Co., Stockton, CA, USA), 160 g of powdered milk were used. For identification purpose, flies were previously (Carnation®, Nestle, USA), 40 g of sodium polyacrylate gel marked with acrylic paint on the thorax with white for 4-day- (Waterlock G-400, Grain Processing Corp, Muscatine, IA, old and red for 8-day-old. Flies also were dissected after USA), and 140 ml of citrated bovine blood (collected from a experiments to determine the ovarian stage. Each experiment local abattoir). These ingredients were mixed with 600 ml of was replicated six times. The positions of the treated and water with 1 ml of 37 % formaldehyde solution (Sigma- control containers were randomized each time. Aldrich, St. Louis, MO, USA) as a fungistat to prepare 1 l of diet. Eggs were collected from 8-day-old gravid females over Oviposition Assays To determine oviposition preference, tests rotten liver, and were placed directly on the diet in a 20×15× were conducted in 30×30×30 cm wire mesh cages as 12 cm rearing tub (~200 mg eggs per liter diet). Pupae were described in the above section. Fifty 8-day-old gravid females collected after 5 days in a 26×20×6 cm tray of vermiculite were transferred from the colony cage to each test cage placed under the rearing tub. Adults emerged from pupae 6 days through the front opening as described. Two disposable Petri after pupation. Flies were maintained on sugar cubes, powdered dishes (10 cm diam) were used to contain the oviposition egg, and honey paste and water. Eight-day-old gravid females substrate test samples. Each Petri dish was lined with a filter were used for the oviposition assay. paperonwhich5gof(a)freshorrottenbeefliver,(b)freshor rotten chicken liver, or (c) rotten beef or rotten chicken liver Livers Beef and chicken livers were purchased from a local were placed. Each pair of Petri dishes then was introduced into grocery store (HyVee Inc. Lincoln, NE, USA) and were each test cage and placed on the base of the cage, one at each Author's personal copy

J Chem Ecol (2013) 39:1407–1414 1409 diagonal corner. Similarly, to test the efficacy of the identified placed on top of the fresh liver, or an equal volume of ethyl chemicals, Petri dishes were introduced into additional test alcohol as control. Flies were given the opportunity to oviposit cages with gravid females as above. These were baited with for 1 hr when the cage was kept in the darkness at 35 °C and fresh chicken liver together with 1 ml attractant solution 60 % R.H. After 1 hr, the Petri dishes were removed from the containing a single or a blend of identified compounds from cage, the egg masses in each dish were collected, and the rotten chicken liver volatiles applied on a dental wick (1 cm) weight of eggs per dish was determined with a digital balance. A total of 6 replicates were conducted for each oviposition assay. The positions of the treated and control plates were A 70 randomized each time. * 60 Collection of Volatiles, Chemical Analysis, and Lure 50 Preparation Solid-phase microextraction (SPME) was used to collect odor from 5 g of fresh or rotten liver placed in a 40 20 ml glass vial. A SPME fiber (70 μm polydimethylsiloxane/

30 carboxen, Supelco, Bellefonte, PA, USA) was preconditioned for 1 hr at 250 °C in the injector of a gas chromatograph (GC). 20 For odor collection, the fiber was exposed approx. 2–3cm Amount of laid eggs (mg) 10 above the liver in the vial for 3 hr at the same temperature (35 °C in a water bath) used for fly oviposition experiments, 0 which gave sufficient time for equilibration of all released Fresh beef liver Rotten beef liver volatiles. The loaded SPME fiber was desorbed in the injection port of a coupled gas chromatography/mass B 180 * spectrometry (GC/MS) system (HP 7890 GC interfaced to 160 an HP 5975 Mass Selective Detector Agilent Technologies, Inc., Santa Clara, CA, USA). The GC was equipped with a 140 FFAP and DB-5 column (30 m×0:25 μm ID, Agilent) with 120 splitless injection (250 °C). The oven temperature program

100 started at 50 °C for 3 min, increased to 170 °C at 5 °C/min, and

80 Fresh beef liver 60 Amount of eggs laid (mg) 40 Acetoin

20 Rotten beef liver

0 acetate Fresh chicken liver Rotten chicken liver Ethyl acetate -Phenethyl -Phenylethanol β Isoamylacetate C 2 Acetic acid 450 Pentanol * 400 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 350 Time (min) Fresh chicken liver 300

250

200

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Amount of eggs laid (mg) 100

50 3-Methyl butanol 3-Methyl

0 disulfideDimethyl Isovaleraldehyde Dimethyl trisulfideDimethyl 4-Methylpentan-2-one Rotten beef liver Rotten chicken liver Fig. 1 Comparisons of egg deposition of gravid female Cochliomyia 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 macellaria on fresh and rotten beef (a) and chicken (b) livers, and Time (min) different rotten livers (c)(Asterisk indicates significant differences by Fig. 2 GC/MS Analyses of volatiles collected by SPME from fresh and Student t-test, P <0.05; N =8) rotten livers Author's personal copy

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Table 1 Compounds released from rotten beef and chicken livers and a dental wick (1 cm). This was placed in 20 ml glass vial their relative ratios determined by solid phase microextraction absorption for 30 min. before initiation of SPME collection of and GC/MS analysis volatiles under the same condition as described for liver Compound Relative Ratio odor collection and analysis by GC/MS. A total of 3 (mean ± SE; N =5–6) replicates was performed. Rotten chicken liver Electroantennogram (EAG) Responses EAG responses were Isovaleraldehyde 28±6 recorded with Ag-AgCl electrodes inserted into glass 4-Methylpentan-2-one 41±15 microelectrodes containing electrogel (Spectro 360, Parker Dimethyl disulfide 37±14 Laboratory, NJ, USA). Signals were amplified with a 3-Methylbutanol 100±0 high-impedance D.C. amplifier with automatic baseline Dimethyl trisulfide 7±2 drift compensation (Syntech Equipment and Research, 2-Phenylethanol 13±6 Kirchzarten, Germany), and data were captured and analyzed p-Cresol 12±9 with EAG-Pro 4.6 (Syntech). The base electrode was inserted Rotten beef liver into the back of the head of an 8-day-old female SSW fly Ethyl acetate 100±0 excised just behind the vertex by using a pair of Vannas Isoamyl acetate 15±3 scissors (World Precision Instruments, Sarasota, FL, USA). 1-Pentanol 15±3 A recording electrode was placed in contact with the tip of the Acetic acid 1.1±0.1 (funicle). 2-Phenylethyl acetate 15±3 Antennal responses to chemical stimuli were recorded to 2-Phenylethanol 6±0.7 10 μg of synthetic standards in 10 μl hexane (B&J high grade solvent, Honeywell, Morristown, NJ, USA) applied to filter- paper strips (0.5×2.0 cm, Whatman No. 1) that were inserted then increased to 240 °C at 15 °C/min. Helium was into Pasteur pipettes (15 cm long). Control puffs (hexane only) used as the carrier gas (3 ml/min). Mass spectra were recorded were applied after each puff of a test stimulus. The order of from 30 to 550 amu with electronic impact ionization at exposure of each stimulus to each antenna was defined 70 eV. Identifications of liver volatiles were confirmed by randomly, and at 1-min intervals for antennal sensitivity comparison of retention indices and mass spectra of authentic recovery. A total of 7–10 antennae of gravid female SSW flies standards that were purchased from Sigma-Aldrich, St. Louis, were tested. Recording time for each antenna lasted less MO, USA, with >95 % purity. than10 min due to the relative short life and stability of the Lures were prepared by impregnating the synthetic excised antenna, and then another freshly cut antenna was attractant blend (0.5 mg total dissolved in 1 ml ethanol) onto used for new recordings.

Fig. 3 Absolute EAG responses from antennae of gravid female Cochliomyia macellaria to volatile compounds identified from rotten chicken liver (10 μg) (N =6–8). Dashed lines inside bars in figures were mean EAG responses to control puffs Author's personal copy

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Table 2 Relative percentages of compounds observed in SPME collections from rotten chicken liver, in loadings on the dental wick lure and in the volatiles released from the lure isoValeraldehyde 4-Methylpentan-2-one Dimethyl disulfide 3-Methylbutanol Dimethyl trisulphide 2-Phenylethanol p-Cresol

Relative percentages in volatiles from rotten chicken liver 28 41 37 100 7 13 12 Relative percentages loaded in lure 12 12 14 100 7 13 12 Relative percentages observed from lure 28 42 37 100 7 13 12

Statistical Analysis The significance of differences from synthetic attractant blends) were determined by multi- individual means (EAG responses among different way ANOVA followed by LSD test (PASW Statistics stimuli, amount of eggs laid from liver, and identified 18, SPSS Inc.) and Student t -test (EAG responses to

Fig. 4 Landing rates of 8-day-old A male and female Cochliomyia 16 Male Female macellaria (a) and young and A gravid female C. macellaria 14 (b) on fresh chicken liver with or without the synthetic 12 7-component blend. Different letters on top of bars indicate significant differences (Student 10 t-test, P <0.05; N =6)

8

B 6 a a

4 Observed number of landings on treatments

2

0 Blend Control

B 16

A 4-d-old 8-d-old 14

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a 6 a

4 Number of landings observed from tested females 2

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1412 J Chem Ecol (2013) 39:1407–1414 control). Results with P <0.05 were considered to be compounds were identified from rotten beef and chicken livers, statistically significant. respectively (Table 1); their structures were confirmed further by comparisons of retention indices and mass spectra with those of authentic compounds. Significant differences were Results found in amounts released from the beef and chicken rotten livers, with only 2-phenylethanol occurring in both. Oviposition on Liver Preparations Oviposition experiments This compound also has been identified in fluid from wounds showed that gravid females laid over 7–10 times more eggs on infested with PSW larvae and elicited electrophysiological rotten livers relative to fresh livers (Fig. 1a and b; t =2.78, responses from PSW antennae (Cork 1994; Cork and Hall P <0.001 for beef liver; t =2.82, P <0.001 for chicken liver). 2007). Furthermore, dimethyl disulfide and p-cresol also Furthermore, over seven times more eggs were laid on are two of the constituent compounds of the swormlure rotten chicken liver than on rotten beef liver (Fig. 1c, t =2.77, bait used for PSW flies (Mackley and Brown 1984). P <0.002). EAG Responses of Gravid Females to Synthetic Analyses of Volatiles from Fresh and Rotten Liver No volatile Compounds Absolute EAG responses were recorded to compounds were detected from either type of fresh liver during the seven compounds from rotten chicken liver (Fig. 3) the 3 hr collection, with exception of a trace of acetoin found as this was preferred to rotten beef liver in oviposition from the fresh beef liver (Fig. 2). Totals of 6 and 7 volatile bioassays (Fig. 1c). Significant EAG responses were elicited

400

350 a

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250 b b

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Egg deposition (mg) deposition Egg c c 150 c

c c c cd cd 100 cd cd

d cd d 50 e

0 B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 S1 S2 S3 S4 S5 S6 S7

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 S1 S2 S3 S4 S5 S6 S7 Isovaleraldehyde + + + + + + + + + 4-Methylpentan-2-one + + Dimethyl disulfide + + + + + + + + 3-Methyl butanol + + Dimethyl trisulfide + + + + + + + + 2-Phenylethanol + + P-Cresol + + + + + + + Fig. 5 OvipositionbygravidfemaleCochliomyia macellaria on fresh chicken livers baited with different combinations of seven synthetic compounds. Different letters on bars indicate significant differences (ANOVA followed by LSD test, P <0.05; N =6) Author's personal copy

J Chem Ecol (2013) 39:1407–1414 1413 from antennae of gravid female SSW to all seven compounds documented (Bishopp 1916, 1937; Guillot et al. 1977a, b; compared to the hexane control (t =2.96–22.6, P <0.05). Krafsur et al. 1979; Laake and Cushing 1930; Lindquist The average EAG response to the control from antennae 1938; 1954; Mackley 1986; Mangan and Thomas 1989; of gravid females was 57±11 μV. There were no significant Spencer et al. 1981). Hall and Doisy (1993) further identified differences in EAG responses to the seven test compounds that SSW were more attractive to beef liver that was decayed (F =1.50, df =6, 72, P >0.05). This suggests that olfactory and over 1 day-old. A synthetic lure for PSW has been sensilla located on the antennae of gravid females may play developed as “swormlure” containing 2-butanol, isobutanol, important roles for oviposition site location. dimethyl disulfide, acetic acid, butyric acid, valeric acid, phenol, p-cresol, benzoic acid and indole (Coppedge et al. Preparation of Lures The ratio detected from the absorption of 1977; Jones et al. 1976;Lindquist1938;Mackley1986). volatiles on the SPME fiber might be different from that released Our study showed that rotten chicken liver is preferred over from the liver due to their different vapor pressures and different rotten beef liver for oviposition by SSW females. Seven affinities of those compounds. The loading ratio of the seven compounds emitted by rotten chicken liver were identified. compounds for the dental wick lure used in oviposition assays A blend of the seven synthetic compounds increased landing was adjusted so that the ratios observed in analyses of volatiles by female SSW on fresh chicken liver and increased collected by SPME from the lure were similar to that previously oviposition. measured from rotten chicken liver (Table 2). The 7-component blend reported here is different from that of swormlure, which attracts both males and females of PSW Landing Response to Oviposition Substrate Significantly as well as SSW in the field (Brown and Mackley 1983; more females than males landed on fresh chicken liver treated Mackley and Brown 1984;Snowetal.1982), having only with the 7-component blend during the 15-min observation, dimethyl disulfide and p-cresol in common. It is possible that whereas, significantly fewer females landed on the unbaited these differences in chemical composition make the present control substrate (Fig. 4a). There was no significant difference blend a female-specific attractant as opposed to a generalized between the number of males landed on treated substrate and attractant for both sexes as in the case of swormlure. Field those landed on the control. Significantly more 8-day-old than studies to test the hypothesis will be conducted when an 4-day-old females landed on the substrate treated with the appropriate location with both SSW and PSW is identified. blend, whereas, there was no significant difference between Although the identified synthetic blend elicited strong the number of 4-day-old and 8-day-old females that landed on attraction and oviposition from female SSW in the laboratory the control substrate (Fig. 4b). These results strongly support assays, the effectiveness should be demonstrated under field that the attractant odorants from the rotten liver are more conditions. It also will be interesting to see if the blend attracts attractive to gravid female SSW than males and the young PSW. This is the first study that reports a synthetic blend as an females. attractant for secondary screwworms, which, with further development might be applied for monitoring and sampling Oviposition Responses of Gravid Females to Synthetic Blends gravid SSW field populations and their associated transmitted and Individual Compounds A synthetic blend of 7 diseases in livestock. components (B1) identified from the rotten chicken liver placed on top of fresh chicken liver caused the most egg deposition with over 300 mg of eggs from the gravid females Acknowledgments We express our gratitude to Dennis Berkebile, tested, compared to less than 20 mg on the unbaited, fresh USDA-ARS, Lincoln, NE for his technical help with this study and Roger liver control (Fig. 5, df =16,85, F =23.03, P <0.001). The Leopold, USDA-ARS, Fargo, ND for supplying the pupae. This work was done in cooperation with the Institute of Agriculture and Natural fresh liver baited with a 4-component blend (B2) and a 3- Resources, University of Nebraska-Lincoln, and supported partly by the component blend (B10) also received over 200 mg eggs. regional project 1030. Other blends tested (B3-B9) elicited less than 150 mg eggs. Among the individual compounds tested, only dimethyl trisulfide resulted in deposition of nearly 100 mg of eggs. The other individual compounds attracted only a few females’ References oviposition and was 30–80 mg of eggs (Fig. 5). Anderson GS, Huitson NR (2004) Myiasis in pet animals in British Columbia: the potential of forensic entomology for determining Discussion duration of possible neglect. Can Vet J 45:993–998 Bishopp FC (1916) Flytraps and their operation. USDA Farmers’ Bull. No. 734, 14 pp, Washington, D.C. Methods of monitoring screwworm populations by using traps Bishopp FC (1937) Flytraps and their operation (revised). USDA baited with rotten meat and sentinel animals are well- Farmers’ Bull. No. 734 (rev.), Washington, D.C. Author's personal copy

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