Comparison of Techniques for Sampling Adult Necrophilous Insects From Pig Carcasses

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1 Smpling, Distriution, Dispersl Journl of Medicl Entomology, XX(X), 8, 8 doi:.93/jme/tjx55 Reserch omprison of Techniques for Smpling dult Necrophilous Insects From Pig rcsses ngel ruise,, Edurdo Htno, Dvid W. Wtson, nd oy Schl,3 Deprtment of Entomology nd Plnt Pthology, North rolin Stte University, mpus ox 73, Rleigh, N , Present ddress: rolin iologicl Supply ompny, 7 York Rod, urlington, N , nd 3 orresponding uthor, e-mil: coy@ncsu.edu Suject Editor: Json yrd Received 5 Octoer 7; Editoril decision Decemer 7 strct Studies of the pre-coloniztion intervl nd mechnisms driving necrophilous insect ecologicl succession depend on effective smpling of dult insects nd knowledge of their diel nd successionl ctivity ptterns. The numer of insects trpped, their diversity, nd diel periodicity were compred with four smpling methods on neonte pigs. Smpling method, time of dy nd decomposition ge of the pigs significntly ffected the numer of insects smpled from pigs. We lso found significnt interctions of smpling method nd decomposition dy, time of smpling nd decomposition dy. No single method ws superior to the other methods during ll three decomposition dys. Smpling times fter noon yielded the lrgest smples during the first d of decomposition. On dy 3 of decomposition however, ll smpling times were eqully effective. Therefore, to mximize insect collections from neonte pigs, the method used to smple must vry y decomposition dy. The suction trp collected the most species-rich smples, ut sticky trp smples were the most diverse, when oth species richness nd evenness were fctored into Shnnon diversity index. Repeted smpling during the noon to 8: hours period ws most effective to otin the mximum diversity of trpped insects. The integrtion of multiple smpling techniques would most effectively smple the necrophilous insect community. However, ecuse ll four tested methods were deficient t smpling eetle species, future work should focus on optimizing the most promising methods, lone or in comintions, nd incorporte hnd-collections of eetles. Key words: iodiversity, ecology nd ehvior, forensic entomology, lliphoride, Srcophgide Forensic entomology links the science of entomology nd the judicil system, most notly in deth investigtions. Forensic investigtors use entomologicl evidence (e.g., insect species nd developmentl stges) from crime scene nd iotic nd iotic chrcteristics of the environment (e.g., plnt cover, soil type, nd temperture) to mke inferences out the time since deth, the postmortem intervl (PMI) (Goff 993). etter understnding of these fctors nd other prmeters, such s geogrphic loction nd level of concelment, my enle the prediction of necrophilous insect species succession on decomposing ody, which is pivotl element in PMI determintion (Tomerlin et l. ). The chrcteriztion of ecologicl succession in generl, nd of necrophilous insects in prticulr, depends on ccurte documenttion of temporl nd sptil chnges in species diversity, richness, nd undnce. To document the complete successionl pttern of insects rriving to ephemerl ecologicl resources, such s smll crcsses tht re rpidly colonized y flying insects, the smpling methods used to collect newly rriving dults must e reltively unised nd esy to implement without disrupting the successionl process. The eril sweep net (sweep net, henceforth) is the most common smpling tool in forensic entomology reserch nd, in comintion with hnd-collections of lrve on nd round the ody, is the pproved technique for investigtors to collect necrophilous insects (yrd et l. ). While this comintion of methods dequtely smples the fun on lrge decomposing odies, it hs proven indequte for succession ecology studies on smll pigs (Schoenly et l. 7). Pigs re the most common model used in forensic studies in plce of humn cdvers ecuse they re esy to cquire nd decompose much like humns (tts nd Goff 99, rvlho et l. ). Medium-sized 3-kg pigs (wened feeder pigs) re most commonly used in such studies, ut reserchers often choose to use smller pigs (tts nd Goff 99, rcher, Zimmermn nd Wllce 8). lthough smller sized pigs hve een shown to dequtely represent the locl species composition dynmics throughout succession, they tend to ttrct fewer insects thn lrger crcsses (Kuusel nd Hnski 98, Hewdikrm nd Goff 99). Smpling smll crcsses, such s neonte pigs, is chllenging ecuse they represent smll trgets nd rriving insects re redily distured y ctive smpling methods like sweep nets nd do not redily return to such The uthor(s) 8. Pulished y Oxford University Press on ehlf of Entomologicl Society of meric. ll rights reserved. For permissions, plese e-mil: journls.permissions@oup.com. Downloded from y D H Hill Lirry - cquis Dept S user on Ferury 8

2 Journl of Medicl Entomology, 8, Vol. XX, No. XX smll ody ( personl oservtions). Nevertheless, neonte pigs re esy to cquire nd much less costly thn lrger pigs, so it is importnt to identify unised nd less disruptive smpling methods for documenting their ssocited fun. Severl pssive smpling techniques, such s ited emergence trps, sticky trps (typiclly ited with odor or tissue), nd pitfll trps, hve een used y reserchers to smple necrophilous insects with fir success (Schoenly 98, shworth nd Wll 99, Hll et l. 3, Schoenly et l. 7, e stro et l. 9). The lck of direct nd quntittive comprisons of ctive nd pssive smpling methods motivted us to compre two ctive nd two pssive pproches to ssess the diversity nd numer of insects trpped ner neonte pigs, s well s the reltive ese of use of these methods. The sweep net ws chosen s one of the ctive methods ecuse of its widespred use in forensic reserch nd prctice. ecuse no other ctive smpling methods could e found in the literture for smpling flying dult necrophilous insects, we dpted suction trp tht trgets flying mosquitoes (Vzquez-Prokopec et l. 9). n emergence trp nd sticky trps were selected s the two pssive methods, with the whole pig itself serving s the lure, unlike severl previous studies tht used only specific tissues or synthetic odor lends (shworth nd Wll 99, Hll et l. 3). Of these methods, only the sweep net is commonly used in the prctice of forensic entomology, oth y investigtors t the crime scene nd y reserchers in the field (Schoenly et l. 5, yrd et l., Goff, Mtuszewski et l. ). To our knowledge, none of the methods used in our investigtion hve ever een pplied to the neonte pig model. dditionlly, we compred the diel pttern of trpping with the four methods to identify the optiml time of dy for smpling to chieve mximum diversity nd undnce. Mterils nd Methods Study Site nd Experimentl Design Experiments were conducted t North rolin Stte University s Lke Wheeler Rod Field L in Rleigh, N (35.793, ). Fully frozen stillorn pigs were cquired from the University s Swine Eductionl Unit. ecuse four smpling methods were compred concurrently, four pigs were used in ech replicte of the experiment, with ech pig ssigned different collection method. Frozen pigs were plced in the field t : hours on the dy of the first smpling (dy ). They were spced 5 m prt in full sunlight nd were smpled four times dily t 9:, : (noon), 5:, nd 8: hours over 3-d period to exmine oth the succession process nd its diel periodicity. The spcing etween pigs ensured experimentl independence of the pigs, nd the full sunlight conditions reduced ny potentil vriility cused y shding or vegettive differences t the four loctions (Shen et l. 993, Shrnowski et l. 8, Perez et l. 5). Freezing the pigs prior to field plcement is not known to ffect the successionl pttern (ugjski et l. ), nd pigs t reched mient temperture in ~.5 h in full sunlight ( personl oservtions). ecuse pigs were frozen within minutes of deth, we re equting decomposition dy nd dys since plcement in the field for ll nlyses. The experiment ws replicted totl of five times during June nd July. Smpling Methods Two ctive nd two pssive insect smpling methods were used. For the purposes of this work, modified Prokopck spirtor (suction trp) (Vzquez-Prokopec et l. 9) nd sweep net were considered ctive collection methods, while n emergence trp nd sticky trps were considered pssive. ll methods in this experiment focused on rriving dult necrophilous insects of orders Dipter nd oleopter. Smpling on ll four pigs ws performed simultneously over -min period. Pssive smpling methods remined uninterrupted for the full -min intervl. Preliminry work reveled, however, tht oth ctive methods were highly disruptive to insects t the pig ody. Therefore, ctive smpling ws performed for two interrupted.5 min intervls on the pigs to llow insects to return to the pigs. Thus, suction trp smpling ws conducted for.5 min on pig #, then sweep net smpling ws conducted on pig #, nd this process ws repeted once during the -min intervl, llowing distured insects to return to the pig. Menwhile, pssive smpling on pig #3 (emergence trp) nd pig # (sticky trps) remined uninterrupted for min. Modified Prokopck spirtor The Prokopck spirtor ws developed s portle, cost-effective wy to smple dult mosquitoes (Vzquez-Prokopec et l. 9). Like the originl design, our suction trp fetured n in-line lower motor, ruer coupling, nd D ttery (Fig. ). Unlike the Prokopck however, our trp ws not ttched to telescoping pole nd did not hve uilt-in collection cup or ckpck. Insted, wire mesh window screening ws ttched to cm long, 8 cm dimeter section of PV. The mesh llowed insects to e cptured nd prevented them from eing pulled into the motor s ldes. The PV trp ws connected to.5 m 3 /min in-line lower (ttwood Turo, Item #77-, Lowell, MI) with -to-8 cm ruer coupling. The lower ws powered y V 3mpHR seled-electrolyte ttery (XTREME, XTXL-S, tteries Plus, ethel, T) with n in-line mnul switch. Smpling with the suction trp ws limited to the re on nd ove the pig nd did not include ny locl vegettion. t the end of ech.5 min smpling intervl, insects were trnsferred into 7% ethnol Sweep Net 38-cm-dimeter sweep net with fine mesh netting ws used to smple necrophilous insects (Fig. D). eril sweeping ws performed in zig zg motion with 8 twists s descried y Wyne nd Wllce (yrd et l. ). s with suction smpling, sweep net smpling ws limited to the re ove the pig nd did not include ny sweeps of locl vegettion. The contents from ech.5 min of ctive sweeping were emptied into 7% ethnol. Emergence Trp 9 mesh, cm soil emergence trp (ugdorm Item #T3, ioquip, Rncho Dominguez, ) ws elevted cm from the ground y stkes nd plced over decomposing pig (Fig. ), which llowed insects to enter the trp. The collection ottle in the emergence trp contined 7% ethnol s the killing gent. This pssive trp ws deployed over the pig for min, fter which it ws removed nd the collection ottle retrieved. Sticky Trps Sticky trps were chosen s pssive method ecuse of their documented use s successful ited trps (Hll et l. 3, ork nd Hll 7). To mximize the surfce re of the sticky trps, they were mounted on wooden frme tht llowed for six unscented insect monitoring sticky trps (LoLine, &G, Jckson, G) to e simultneously plced t severl heights round the pig (Fig. ). To llow for esy plcement nd removl of the trps, cork stoppers were ttched to the wooden frme with wood glue t vrious points, nd trps were ttched with stndrd pushpins. The frme Downloded from y D H Hill Lirry - cquis Dept S user on Ferury 8

3 Journl of Medicl Entomology, 8, Vol. XX, No. XX 3 Fig.. Trpping methods used in this study. () Modified Prokopck spirtor. () Emergence trp elevted cm off the ground to ensure insect rrivl ws not interrupted. () Six sticky trps on wooden frme tht ws plced over the pig lengthwise. (D) eril sweep net. In (), ) 8-cm-dimeter PV; ) -to-8 cm ruer coupling; 3) in-line lower motor; ) in-line on/off switch; nd 5) V 3mpHR ttery. with six sticky trps ws plced over the pig nd left undistured for min. The trps were collected in plstic cling wrp. Identifictions Orders Dipter nd oleopter were the min smpling trgets, with emphsis on those necrophilous fmilies commonly used in forensic entomology for PMI determintions. Diptern fmilies were identified using keys in Whitworth (), nd oleoptern fmilies were identified with Triplehorn et l. (5) nd lmeid nd Mise (9). ecuse of their significnt role s primry colonizers of decomposing odies, the low flies were further identified to species level using Whitworth (). Some specimens were identified t N Stte s Plnt Disese nd Insect linic y Dr. Mtthew ertone. Insects on sticky trps were identified in situ. nlysis Smpling method, time of dy, nd dy of decomposition were recorded for ll smples. Sttisticl nlyses were performed in R, version 3.. (R Development ore Tem ). Totl counts of insects were nlyzed to ssess the effect of smpling method, time of dy nd decomposition dy with negtive inomil generlized liner mixed-effect models (N glmm) with experimentl locks nd individul pigs s rndom effects using the lme pckge (tes et l. 5). P-vlues of comprisons etween tretment levels were clculted sed on the z-distriution. Significnce of ech vrile in the model ws ssessed y compring models with nd without respective vriles or interctions using χ test (rwley ). Diversity y method ws ssessed through overll counts y method, reltive percentge of ech txon y method nd time, nd with three ecologicl indices (richness, evenness, nd Shnnon diversity). This llowed us to ssess whether there were smpling time (diel effects) or smpling method effects on the txonomic composition of smples. When considering diversity y smpling method, we sudivided diversity into two components: species richness nd species evenness (lrke nd Wrwick 99). Species richness refers to the numer of forensiclly relevnt tx collected respectively y ech of the four smpling methods (Spellererg nd Fedor 3). Species richness (S) ws represented y the numer of tx tht were collected y ech smpling method cross the five replictes. Species evenness (J ) is the consistency in the numer of individuls cross tx. We clculted richness nd evenness indices, s well s Shnnon diversity index, which considers oth richness nd evenness s fctors in its clcultion (lrke nd Wrwick 99, Schüpch et l. ). For the species evenness index nd the Shnnon diversity index, we included ll tx smpled within the community. Therefore, tx tht were not trpped t ll y some methods were represented y zero. Results Min Effects: Smpling Method, Smpling Time, nd Decomposition ge ll three min effects significntly ffected the numer of insects trpped (Tle ), nd these ptterns re shown in Fig.. Only some of their interctions, however, were significnt (Tle ), nd these re Downloded from y D H Hill Lirry - cquis Dept S user on Ferury 8

4 Journl of Medicl Entomology, 8, Vol. XX, No. XX Tle. Test sttistics of model comprisons fter stepwise removl of interctions nd vriles Model Removed term χ df P (Sturted model) Method:Time:Dy Method:Time Dy:Time 7.5 <. Method:Dy 39.5 <. Dy 9.35 <. Time.87 3 <. Method. 3 <. Sttisticl nlysis ws performed using negtive inomil generlized liner mixed model nd comprison etween models ws performed using χ test (see Mteril nd Methods for detils). Models were compred to the immedite preceding model following the steps in model simplifiction. χ = hisqure vlue; df = degrees of freedom used y ech model; P = P-vlues of model comprisons. Order of stepwise model simplifiction. Interctions or vrile removed from preceding model. Method = smpling method; Time = smpling time of dy; Dy = dy of decomposition. shown in Fig. 3. The emergence trp collected significntly fewer insects thn the other three methods (Fig. ). There were no significnt differences in the numers of insects smpled y the suction trp, sweep net, nd sticky trps; these methods were eqully effective in terms of the totl insects smpled, disregrding their txonomic ffilitions. Smpling counts cross ll methods incresed s the dy progressed (Fig. ). Insect counts t 8: hours were significntly higher thn t ny other smpling time. The dy of decomposition lso ffected the overll numer of insects smpled (Fig. ) with significnt differences mong ll 3 d of decomposition. Effect of Decomposition Dy nd Smpling Time The totl numer of insects collected cross ll methods ws significntly ffected y the interction of smpling time nd decomposition dy (N glmm, Tle, Fig. 3). During the first d of decomposition, fternoon smpling resulted in significntly lrger smples thn smpling t or efore noon. On the second dy of decomposition, this effect ws continuous, with the numer of insects trpped significntly incresing t ech smpling time. The 8: hours smpling time on this dy collected the highest numer of insects from ny smpling time or dy of decomposition. It is importnt to note tht pigs were thwing on dy of decomposition, so the ineffectiveness of erly smpling times on this dy my relte to the slow decomposition of the pigs. Refrigertion is not known to lter the rrivl times of insects, ut the effect of freezing (without thwing period) is unknown (ugjski et l. ). On the third dy of decomposition, the time t which smpling ws performed did not ffect the overll numer of insects trpped cross methods; ll four smpling times were eqully effective for trpping insects. Effect of Smpling Method nd Decomposition Dy Dy of decomposition hd significnt effect on the numer of insects trpped y method (N glmm, Tle, Fig. 3). ll smpling methods collected their lowest respective numer of insects on dy of decomposition. On this dy, the sweep net nd sticky trp methods were most effective, lthough the totl numers of insects smpled y these two methods were significntly lower thn for ny other dy. The distinction mong methods ws less ovious on dy of decomposition, when the suction trp, sweep net, nd sticky trps Insects smpled per min Insects smpled per min Insects smpled per min Emergence were eqully effective for smpling insects. y dy 3 of decomposition, cler differences emerged mong the methods, with the suction trp smpling more thn twice s mny insects s ny other method. The smpling method most influenced y decomposition dy ws the suction trp. On decomposition dy, the suction nd emergence trps smpled the fewest insects. y dy of decomposition, Sticky Sweep net Suction Smpling method 5 9: : 5: 8: Smpling time Decomposition dy Fig.. Numer of insects smpled, showing min effects of () ech smpling method (N glmm, χ =., df = 3, P <.), () smpling time of dy (N glmm, χ =.87, df = 3, P <.), nd () decomposition dy (N glmm, χ = 9.35, df =, P <.). rs show men vlues ± SE. Numers within the rs denote numer of min smpling sessions. rs leled with the sme letter re not significntly different (P <.5). c c Downloded from y D H Hill Lirry - cquis Dept S user on Ferury 8

5 Journl of Medicl Entomology, 8, Vol. XX, No. XX 5 Insects smpled per min Insects smpled per min 8 8 Dy Dy Dy 3 the suction trp, sweep net, nd sticky trp were eqully effective. On dy 3 however, the suction trp significntly surpssed ny of the other methods for the numer of insects trpped. ll other methods remined stedy in their rnkings on dys nd 3, wheres the efficcy of the suction trp incresed on ech successive dy. Txonomic Ptterns y Smpling Method Only forensiclly relevnt insects of the Orders Dipter nd oleopter were included in the nlyses, nd clliphorids were further identified to species. Our collections included seven clliphorid species (Fig. ) tht overlpped with collections y mmck et l. (), who lso smpled in the sme loction. Insect counts were compred cross smpling methods (Fig. ) nd y the reltive representtion of ech txon y ech of the four smpling methods (Fig. ). lliphoride ws the most undnt fmily trpped, followed y Srcophgide (Fig. ). Srcophgid flies nd the four most undnt species of clliphorid flies collected c d 9: : 5: 8: Smpling time Emergence Sticky Sweep net Suction Smpling method Fig. 3. Numer of insects smpled, showing only significnt interction effects of () smpling time y decomposition dy (N glmm, χ = 7.5, df =, P <.), nd () smpling method y decomposition dy (N glmm, χ = 39.5, df =, P <.). Other interctions (smpling method y smpling time; smpling time y decomposition dy; nd smpling method y smpling time y decomposition dy) were not significnt (Tle ). rs show men vlues ± SE. In (), rs leled with the sme cpitl nd lower cse letters, respectively, re not significntly different within the sme hour nd dy (P <.5). In (), rs leled with the sme cpitl nd lower cse letters, respectively, re not significntly different within the sme smpling method nd dy (P <.5). c (Lucili illustris Meigen, Phormi regin Meigen, Lucili coeruleiviridis Mcqurt, ochliomyi mcellri F.), were trpped y ll four smpling methods, ut few eetles were trpped y ech of the four methods (Fig. ). Species richness ws highest with the suction trp, with totl of 3 tx trpped, followed y sticky trps ( tx). The suction method collected t lest one insect from ech txon, with the exception of dermestid nd stphylinid eetles (Fig. ). It ws the only method tht cptured Lucili cuprin Wiedemnn nd hrysomy megcephl F., which were the rrest clliphorid species smpled overll (Fig. ). dditionlly, the suction trp ws the only method tht collected Phneus vindex Mcley (Screide) nd Necrophil mericn L. (Silphide). The trditionl sweep net method cptured eight tx, including ll clliphorids except the two rrest species; ut it only cptured representtives of one eetle fmily, Histeride. The emergence trp cptured only five tx nd filed to cpture the three lest undnt clliphorid species nd ny eetles; the emergence trp ws lso the only method tht did not smple Musc domestic L. Sticky trps cptured ll clliphorid species except the two rrest, nd these trps lso cptured species in three eetle fmilies. Sticky trps were the only method tht smpled stphylinid eetles, which, long with histerid eetles, were the most undnt eetle fmilies collected overll cross ll methods. Overll, none of the methods collected ll the eetle tx, nd only the suction trp smpled ll fly tx (Fig. ). Txonomic Ptterns y Diel Periodicity No tx were unique to the 9: hours smpling time, ut severl tx were unique to ech of the other smpling times (dt not shown). Dermestid eetles nd. megcephl, rre low fly in this study, were trpped only t : hours. Lucili cuprin, nother rre low fly in this study, ws trpped only t 5: hours, nd N. mericn, rre silphid eetle, ws trpped only t 8: hours. Severl other tx were trpped during two of the four smpling times, including histerid eetles t : nd 5: hours, Lucili serict Meigen t : nd 8: hours, nd scr eetles t 5: nd 8: hours. Musc domestic ws trpped t ll smpling times except 9: hours. Diversity Indices Species evenness is mesure of ech species numericl representtion in the community. Given the reltively smll numer of insects trpped per species, with some species not represented t ll in some smpling methods, this index my e strongly ffected y oth highly nd poorly represented species. Indeed, the emergence trp, which hd the lowest txonomic representtion hd the highest evenness index (J =.78). The reltive percentges of ech txon smpled y method re shown in Fig.. The Shnnon index is n overll diversity index tht considers oth species evenness nd species richness, with lrger vlues representing greter diversity. Shnnon index vlues indicted tht the sticky trp collections were the most diverse (H =.78), followed y the suction trp (H =.7). Emergence trp collections were the lest diverse (H =.58). Discussion Ntionl Reserch ouncil report critiqued vrious forensic disciplines, recommending the need for more sound sic iologicl reserch to strengthen foundtionl disciplines nd increse the crediility of findings in the court system (Ntionl Reserch ouncil 9). In response, reserch in forensic entomology hs moved towrd ecologicl nd evolutionry genetics frmeworks Downloded from y D H Hill Lirry - cquis Dept S user on Ferury 8

6 Journl of Medicl Entomology, 8, Vol. XX, No. XX oleopter Phneus vindex (Screide) Necrophil mericn (Silphide) Dermestide Histeride Emergence cky Sweep net Su on Stpylinide Musc domestic (Muscide) 9 Srcophgide 38 hrysomy megcephl Dipter ochliomyi mcellri Phormi regin Lucili cuprin 88 Lucili serict Lucili coeruleiviridis 79 Lucili illustris 85 Totl insects smpled % insects smpled Fig.. Representtion of ech necrophilous insect txon cross ll replictes. () Totl undnce of ll tx (note log scle). () Reltive representtion of ech txon smpled y ech of the four smpling methods. Dipterns without fmily noted re ll lliphoride. The five most undnt tx (L. illustris, P. regin, Srcophgide, L. coeruleiviridis, nd. mcellri) were smpled with ll four methods. Severl rre tx, including L. cuprin,. megcephl, N. mericn, nd Dermestide, were trpped only with the suction method. Stphylinid eetles were only smpled with the sticky trps. (Tomerlin et l., Tomerlin et l. ). Investigtions of the mechnisms driving the ttrction of necrophilous insects to crrion, ecologicl succession, nd other spects of the pre-coloniztion intervl, or the time from deth to the first insect coloniztion, hve een highlighted s prticulrly lcking (Tomerlin et l. ). In ddition, there is need to etter document ecologicl succession ptterns nd growth rtes of necrophilous insects in vrious geogrphic loctions to gin etter understnding of their ecologicl vrition (Souz et l. 997, Turchetto nd Vnin, mendt et l. ). Pivotl to ll these endevors is the efficient, unised, nd representtive smpling of the necrophilous insect community. Smpling Methods The emergence trp smpled significntly fewer insects thn the other three methods, oth in n overll nlysis of ll smpling methods nd in ech of the three decomposition dys. This smpling method should not e used when lrge insect smples re required over short smpling intervls. The other three methods the suction trp, sweep net, nd sticky trp displyed similr efficcy when the totl numers of insects were considered cross ll decomposition dys nd times of dy (Fig. ). However, the reltive efficcy of these three methods vried significntly y decomposition dy, rough pproximtion of PMI. Thus, experiments designed to mximize dily insect collections during ecologicl succession would need to integrte severl complementry methods. oth the eril sweep net nd sticky trp were most effective on the first dy of decomposition, when the decomposing pigs ttrcted reltively few insects (Fig. 3). Of these, the sweep net ws considerly esier to use ecuse the sticky trp design required constructing frme, the use of inconvenient sticky glue, nd retrievl of insects from the trps without dmging them ws difficult. The two ctive methods nd sticky trps were eqully effective on dy, ut the suction trp ws clerly superior on dy 3, when the decomposing odies ttrcted the lrgest numers of insects. The suction trp nd sticky trp methods outcompeted the other smpling methods in representing the gretest diversity of the necrophilous insect community. The suction trp ws the only smpling method tht trpped ll nine fly tx in this study (Fig. ). Flies re the most numerous insects on decomposing odies nd re commonly used in PMI estimtes, so ccurtely documenting their diversity, nd especilly first rrivl on the ody, is of prticulr importnce (tts nd Goff 99, Goff 993). The sweep net nd sticky trps filed to trp the two rrest fly species (L. cuprin nd. megcephl), indicting tht perhps these methods could e improved the sweep net y ltering the motion so tht it is less disruptive, nd the sticky trps y etter utilizing the pig s decomposition odors, known fctors in low fly host finding (shworth nd Wll 99, Stensmyr et l. ). eetle diversity ws criticlly under-represented. ll four smpling methods collected few eetles, yet mny eetles were oserved on the crcsses. The suction trp smpled the gretest eetle diversity, three of the five tx, with the sticky trp nd sweep net collecting two of the five eetle tx, including Stphylinide nd Dermestide, respectively. The insufficiency of ll selected methods for trpping eetles echoes Schoenly et l. s (7) findings when ssessing vrious methods on lrger, 3 7 kg pigs eril nd sticky trp smpling mostly trget flies, with hnd collections or pitfll trps representing etter methods for smpling eetles. Either hnd collections or pitfll trps should e included in Downloded from y D H Hill Lirry - cquis Dept S user on Ferury 8

7 Journl of Medicl Entomology, 8, Vol. XX, No. XX 7 smpling protocol. Nevertheless, severl of our ttempts to use pitfll trps filed ecuse the soil ws highly compcted, requiring disproportionte lor reltive to other smpling methods to smple the pigs. Txonomic identifictions of mny necrophilous insects, especilly flies, depend on setl nd ntennl chrcteristics. From prcticl perspective, it is importnt to note tht the two most promising methods for documenting diversity, the suction trp nd sticky trps, either severely dmged the insect specimens or were cumersome, s noted previously for sticky trps (Nelder et l. 8). Wings nd ntenne were frequently dmged in the suction trp smples, especilly in smll insects tht often were not well retined y the screen mesh. Design modifictions however, such s soft mesh nylon stockings s the collection vessel, should remedy these shortcomings of the suction trp. lthough insects could not redily e removed from sticky trps despite severl ttempts with vrious oils nd solvents, insects remined whole nd could e identified in situ. lmost ll flies lnded on the trp such tht chrcteristic sete on the wings nd ody were undmged nd visile. If insects do not need to e removed or DN-sed identifictions re implemented, the use of sticky trps is not prolemtic, lthough these trps re cumersome to trnsport nd store. Nevertheless, mjor dvntge of the sticky trps is tht they cn smple insects for longer durtions, nd collections cn e redily compred etween trils or experimenters ecuse experimenter is is miniml. Overll, the suction trp outcompeted the other methods for smpling insect diversity, ut it, the sweep net, nd sticky trps smpled complementry tx. oth ctive methods were disruptive to insects on the pig ody, so multiple interrupted smpling sessions were required to ensure tht ll trget insects were cptured. Diel Periodicity In generl, the undnce of necrophilous insects incresed with time of dy, so smpling lter in the dy (i.e., fter noon) yielded lrger smples thn in the morning (Fig. ). This ws prticulrly pprent on dy of decomposition, when fourfold more insects were smpled t 8: hours thn t 5: hours. On dy 3 however, ll smpling times were sttisticlly equivlent in terms of numer of insects trpped. In repeted smpling design with consistent smpling time ech dy, fter noon smpling should e performed, s it will ensure the lrgest smples cross decomposition dys. Such design, however, only trgets lrge smples nd does not consider diversity of smples. Identifying n idel smpling time, when oth overll undnce nd diversity re mximized, depends lrgely on the ims of the experimentl design. We used reltively short -min smpling intervls in our design, so the ltest smpling time, 8: hours, ws fesile. This my not e possile in experiments tht use longer smpling durtions. Nevertheless, our results highlight tht lrge numers of insects re trpped ner dusk (sunset during experiments ~9:3 hours), even though mny species re not ctive t night (Wooldridge et l. 7, Zurwski et l. 9). The length of the smpling intervl nd seson should e considered when smpling lte in the dy. While the fternoon times yielded lrger overll smples, specific tx vried gretly with time. The noon smpling time ws the only period during which dermestid eetles nd. megcephl, rre low fly in this experiment, were cptured, even though noon smples were reltively smll. Other rre species, including L. cuprin nd N. mericn were collected only t 5: nd 8: hours, respectively. Therefore, it is impossile to identify one idel smpling time tht will cpture the diversity nd undnce of the necrophilous insect community. Repeted smpling during the intervl from noon to 8: hours, however, would ccount for ll the oserved diversity, s no unique species were found efore noon. Repeted smpling of communities is pivotl strtegy to infer relile estimtes of ecologicl indices nd succession. The gol of this work ws to identify smpling methods tht would result in lrge nd species rich smples of necrophilous insects from neonte pig crcsses. We found significnt differences in the numer of insects trpped y method, time of dy, nd dy of decomposition. Not surprisingly, comintion of smpling methods deployed for long smpling durtions would pper to e most desirle. However, most studies re constrined y limited work force nd time. Moreover, smpling for long durtions might interfere with the decomposition process nd ecologicl succession. Therefore, severl generl oservtions nd recommendtions emerged from our nlysis:. Studies seeking to mximize the undnce of necrophilous insects over decomposition should smple in lte fternoon (in mid-summer), ut comintion of smpling methods should e used to smple cross dys of decomposition ecuse the effectiveness of smpling methods chnges over time. In ecologicl succession studies tht focus on diversity over time, the suction trp should e used ech dy, ut smpling should e repeted severl times etween noon nd dusk. When lor is limited, the sticky trps might perform well t representing the locl diversity, ut sticky trps re difficult to store, nd it is difficult to isolte undmged voucher specimens without dmge. Nevertheless, in comintion with periodic use of the suction trp or sweep net, this smpling design would e effective.. Our sticky trp design required multiple trps to surround the pig ecuse necrophilous insects re dept t voiding trps s they nvigte to the decomposing pig. n lterntive pproch might e to reduce trp numer, hndling time, nd storge cpcity y directing the pig odors over single sticky trp. Indeed, this pproch is highly effective (ruise 7), ut requires mnipultion of the pig crcss nd therefore is limited to smll pigs. 3. The sweep net, the estlished methodology for smpling lrge pigs, ws effective t smpling insects. Notly however, it ws inconsistent t representing the locl species diversity cross the decomposition process. The need to smple for mximl diversity is highlighted y the oserved significnce of individul species in the decomposition process, independent of their undnce (rippen nd Singh 5). With severl modifictions, s discussed ove, the suction trp, which cptured the gretest species richness from the pigs, might e more effective thn the forensic stndrd, the sweep net.. Smpling of eetles ws reltively ineffective in this study. n importnt gol of future work should e to improve smpling of eetles. oth hnd-collections nd pitfll trps (ctive nd pssive smpling methods, respectively) re lor-intensive. Znetti et l. () found tht comining oth methods yielded undnt eetle smples with less is for certin tx over others. Perhps ground-touching sticky trps should e considered (ruise 7). cknowledgments We would like to thnk the North rolin Stte University Lke Wheeler Field L Swine Unit for providing the neonte pigs, Rick Sntngelo (N Stte) for his invlule help with prepring for these experiments, Dr. Emily Griffith (N Stte) for ssistnce with sttisticl modeling nd nlysis, nd Dr. Mtthew ertone (N Stte) for ssistnce with txonomic identifictions. Prtil funding for this work ws received from Grdute Reserch Fellowship from the Ntionl Science Foundtion (GRFP wrd DGE- 537) nd the lnton J. Whitmire Endowment t N Stte. 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8 8 Journl of Medicl Entomology, 8, Vol. XX, No. XX References ited lmeid, L. M., nd K. M. Mise. 9. Dignosis nd key of the min fmilies nd species of South mericn oleopter of forensic importnce. Rev. rs. Entomol. 53: 7. mendt, J.,. S. Richrds,. P. mposso, R. Zehner, nd M. J. Hll.. Forensic entomology: pplictions nd limittions. Forensic Sci. Med. Pthol. 7: rcher, M.. nnul vrition in rrivl nd deprture times of crrion insects t crcsses: implictions for succession studies in forensic entomology. ust. J. Zool. 5: shworth, J. R., nd R. Wll. 99. Responses of the sheep lowflies Lucili serict nd L. cuprin to odour nd the development of semiochemicl its. Med. Vet. Entomol. 8: tes, D., M. Mchler,. M. olker, nd S.. Wlker. 5. Fitting liner mixed-effects models using lme. J. Stt. Softw. 7: 8. ugjski, K. N.,.. Seddon, nd R. E. Willims.. comprison of low fly (Dipter: lliphoride) nd eetle (oleopter) ctivity on refrigerted only versus frozen-thwed pig crcsses in Indin. J. Med. Entomol. 8: yrd, J. H., W. D. Lord, J. R. Wllce, nd J. K. Tomerlin.. ollection of entomologicl evidence during legl investigtions, pp In J. H. yrd nd J. L. stner (eds.), Forensic entomology: the utility of rthropods in legl investigtions, nd ed. R Press, oc Rton, FL. mmck, J..,.. ohen, K. L. Kreitlow, R. M. Roe, nd D. W. Wtson.. Decomposition of conceled nd exposed porcine remins in the North rolin Piedmont. J. Med. Entomol. 53: rvlho, L. M., P. J. Thyssen,. X. Linhres, nd F.. Plhres.. checklist of rthropods ssocited with pig crrion nd humn corpses in Southestern rzil. Mem. Inst. Oswldo ruz. 95: tts, E. P., nd M. L. Goff. 99. Forensic entomology in criminl investigtions. nnu. Rev. Entomol. 37: lrke, K., nd R. Wrwick. 99. hnge in mrine communities: n pproch to sttisticl nlysis nd interprettion, nd ed. Plymouth Mrine Lortory, Plymouth, UK. ork,., nd M. J. Hll. 7. Development of n odour-ited trget for femle New World screwworm, ochliomyi hominivorx: studies with host its nd synthetic wound fluids. Med. Vet. Entomol. : rwley, M. J.. The R ook. John Wiley & Sons, West Sussex, United Kingdom. rippen, T. L., nd. Singh. 5. Forensic nd decomposition microiology, pp. 9. In J. K. Tomerlin nd M. E. enow (eds.), Forensic entomology: interntionl dimensions nd frontiers. R Press, oc Rton, FL. ruise,. 7. Smpling nd ecologicl succession of dult necrophilous insects. Ph.D. Disserttion, p. 75. North rolin Stte University, Rleigh, North rolin. e stro,. P., D. hichorro,. Serrno, nd M. D. Grcí. 9. modified version of Schoenly trp for collecting srcosprophgous rthropods. Detiled plns nd construction. nles de iologí 3:. Goff, M. L Estimtion of postmortem intervl using rthropod development nd successionl ptterns. Forensic Sci. Rev. 5: 8 9. Goff, M. L.. Forensic entomology, pp In. Mozyni nd. Nozigli (eds.), The forensic lortory hndook procedures nd prctice. Humn Press, New York, NY. Hll, M. J., R.. Hutchinson, R. Frks, Z. J. dms, nd N. P. Wytt. 3. comprison of Lucitrps nd sticky trgets for smpling the lowfly Lucili serict. Med. Vet. Entomol. 7: Hewdikrm, K.., nd M. L. Goff. 99. Effect of crcss size on rte of decomposition nd rthropod succession ptterns. m. J. Forensic Med. Pthol. : 35. Kuusel, S., nd I. Hnski. 98. The structure of crrion fly communities: the size nd the type of crrion. Ecogrphy 5: Mtuszewski, S., M. Szfłowicz, nd. Grzywcz.. Temperturedependent ppernce of forensiclly useful flies on crcsses. Int. J. Legl Med. 8: 3. Ntionl Reserch ouncil. 9. Strengthening Forensic Science in the United Sttes: Pth Forwrd. Wshington, D: The Ntionl cdemies Press. Nelder, M. P., J. W. Mcredie, nd. S. Mjor. 8. low flies visiting decying lligtors: is succession synchronous or synchronous? Psyche J. Entom. 9: Perez,., N. Hskell, nd J. Wells. 5. ommonly used intercrcss distnces pper to e sufficient to ensure independence of crrion insect succession pttern. nn. Entomol. Soc. m. 9: 7 8. R Development ore Tem. R: lnguge nd environment for sttisticl computing computer progrm, version 3... R Development ore Tem, Vienn, ustri. Schoenly, K. 98. Demogrphic it trp. Environ. Entomol. : 5 7. Schoenly, K. G., N. H. Hskell, R. D. Hll, nd S.. Shhid. 5. Does crcss enrichment lter community structure of predceous nd prsitic rthropods? second test of the rthropod sturtion hypothesis t the nthropology Reserch Fcility in Knoxville, Tennessee. J. Forensic Sci. 5: JFS Schoenly, K. G., N. H. Hskell, R. D. Hll, nd J. R. Gur. 7. omprtive performnce nd complementrity of four smpling methods nd rthropod preference tests from humn nd porcine remins t the Forensic nthropology enter in Knoxville, Tennessee. J. Med. Entomol. : Schüpch,., X. Junge, P. Lindemnn-Mtthies, nd T. Wlter.. Sesonlity, diversity nd esthetic vlution of lndscpe plots: n integrtive pproch to ssess lndscpe qulity on different scles. Lnd Use Policy 53: Shrnowski,. J., E. G. Wlker, nd G. S. nderson. 8. Insect succession nd decomposition ptterns on shded nd sunlit crrion in Ssktchewn in three different sesons. Forensic Sci. Int. 79: 9. Shen,. S., L. Messinger, nd M. Ppworth Oservtions of differentil decomposition on sun exposed v. shded pig crrion in costl Wshington Stte. J. Forensic Sci. 38: Souz,., M. De, nd. X. Linhres Dipter nd oleopter of potentil forensic importnce in southestern rzil: reltive undnce nd sesonlity. Med. Vet. Entomol. : 8. Spellererg, I. F., nd P. J. Fedor. 3. triute to lude Shnnon (9 ) nd ple for more rigorous use of species richness, species diversity nd the Shnnon Wiener Index. Glol Ecol. iogeogr. : Stensmyr, M.., I. Urru, I. ollu, M. elnder,. S. Hnsson, nd. M. ngioy.. Pollintion: rotting smell of ded-horse rum florets. Nture. : 5. Tomerlin, J. K., R. Mohr, M. E. enow,. M. Trone, nd S. VnLerhoven.. rodmp for ridging sic nd pplied reserch in forensic entomology. nnu. Rev. Entomol. 5:. Tomerlin, J. K., M. E. enow,. M. Trone, nd R. M. Mohr.. sic reserch in evolution nd ecology enhnces forensics. Trends Ecol. Evol. : Triplehorn,.. J., N. F. orror, D. J... Triplehorn, nd N. F. Johnson. 5. orror nd DeLong s introduction to the study of insects, th ed. Thompson rooks/ole, elmont,. Turchetto, M., nd S. Vnin.. Forensic entomology nd climtic chnge. Forensic Sci. Int. : S7 S9. Vzquez-Prokopec, G. M., W.. Glvin, R. Kelly, nd U. Kitron. 9. new, cost-effective, ttery-powered spirtor for dult mosquito collections. J. Med. Entomol. : Whitworth, T.. Keys to the gener nd species of low flies (Dipter: lliphoride) of meric North of Mexico. Proc. Entomol. Soc. Wsh. 8: Wooldridge, J., L. Scrse, nd R. Wll. 7. Flight ctivity of the lowflies, lliphor vomitori nd Lucili serict, in the drk. Forensic Sci. Int. 7: Znetti, N. I., R. min, E.. Viscirelli, nd N. D. enteno.. ctive serch on crcsses versus pitfll trps: comprison of smpling methods. Neotrop. Entomol. 5:. Zimmermn, K.., nd J. R. Wllce. 8. The potentil to determine postmortem sumersion intervl sed on lgl/ditom diversity on decomposing mmmlin crcsses in rckish ponds in Delwre. J. Forensic Sci. 53: Zurwski, K. N., M. E. enow, J. R. Miller, nd R. W. Merritt. 9. Exmintion of nocturnl low fly (Dipter: lliphoride) oviposition on pig crcsses in mid-michign. J. Med. Entomol. : Downloded from y D H Hill Lirry - cquis Dept S user on Ferury 8