Journal of Science: Vol. 14 | Article 77 Farinha et al.

Small bait traps as accurate predictors of dipteran early colonizers in forensic studies

Ana Farinha1,2a*, Catarina G. Dourado1,2b, Neiva Centeio1c, Ana Rita Oliveira1,2d, Deodália Dias1,2e, and Maria Teresa Rebelo1,2f

1University of Lisbon, Faculty of Sciences, 1749-016 Lisbon, Portugal 2CESAM - Centre for Environmental and Marine Studies, Lisbon, Portugal

Abstract Insect carrion communities vary among habitats and over time. Concerning the dipteran early colonizers of carrion, the use of small bait traps should be accurate because the odors emitted from meat baits should contain many of the volatile organic compounds emitted from the freshly dead mammals. In addition, this kind of trap is easy to replicate and set in position in a given habitat. In the present study, small bait preferences of early Diptera carrion colonizers were examined in an urban biotope. Specifically, three baits were compared (pork muscle, pork liver, and fish flavored cat food) in respect to the number of specimens and species captured and the presence or absence of oviposition at high and low environmental temperatures. A total of 2371 specimens were trapped, primarily belonging to three insect orders, Diptera, Coleoptera, and Hymenoptera. Diptera was the predominant order, with blowflies (Calliphoridae) being the most representative family, followed by filth (). The pork muscle bait was responsible for the highest number of captures and the highest diversity. The community of Diptera collected with the most efficient bait, pork muscle, was compared with the carrion communities reported in the literature from the Iberian Peninsula. Similar taxonomic species composition was found regarding Calliphoridae species. A specimen from all species morphologically identified were also identified at a molecular level using the cytochrome c oxidase I (COI) barcode region, and the sequences were submitted to online databases.

Keywords: bait attraction, cytochrome c oxidase I, DNA barcoding, forensic entomology Abbreviations: CS, Sørensen similarity coefficient; COI, cytochrome c oxidase I Correspondence: a [email protected], b [email protected], c [email protected], d [email protected], e [email protected], f [email protected], *Corresponding author Editor: Tugrul Giray was editor of this paper. Received: 4 January 2013 Accepted: 6 July 2013 Published: 30 May 2014 Copyright: This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed. ISSN: 1536-2442 | Vol. 14, Number 77

Cite this paper as: Farinha A, Dourado CG, Centeio N, Oliveira AR, Dias D, Rebelo MT. 2014. Small bait traps as accurate predictors of dipteran early colonizers in forensic studies. Journal of Insect Science 14(77). Available online: http://www.insectscience.org/14.77

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Journal of Insect Science: Vol. 14 | Article 77 Farinha et al. Introduction Gruner et al. 2007; Brundage et al. 2011; George et al 2013). Flies are typically the first to colonize decomposing remains. Several studies have An accurate identification of specimens is the indicated that the primary important carrion first essential step in forensic studies. Mor- species belong to a relatively small number of phologic methods are usually used (García- families, namely, Calliphoridae, Muscidae, Rojo 2004; Hwang and Turner 2005; Baz et Sarcophagidae, and Fanniidae (Amendt et al. al. 2007; Matuszewski et al. 2010; Anton et al. 2004; Eberhardt and Elliot 2008; Battán 2011; Prado e Castro et al. 2011, 2012), but Horenstein and Salvo 2012), but it is im- these methodologies require available identi- portant to notice that carrion communities fication keys, specialized taxonomic vary both geographically and seasonally (Ar- knowledge, and are time consuming. Thus, naldos et al. 2001; Campobasso et al. 2001; molecular techniques can be of great help in Amendt et al. 2004; Hwang and Turner 2005; identifying specimens. The mitochondrial cy- Anton et al. 2011; Brundage et al. 2011). The tochrome c oxidase subunit I (COI) gene is validation of reference data collected in dif- well established for this purpose, with refer- ferent locations, even within the same ence sequences available for a large number geographic area, has received some attention of species (Amendt et al. 2011). Concerning because variation between habitats, particular- Portugal, only a few molecular studies involv- ly temperature and vegetation, is known to ing the abundant Diptera species with forensic influence insect succession patterns and alter interest have been performed (Cainé et al. decomposition rates (Mann et al. 1990; Tur- 2006, 2009; Ferreira et al. 2011; Oliveira et al. chetto and Vanin 2004; Hwang and Turner 2011; Rolo et al. 2013). Thus, there is still a 2005; Bonacci et al. 2009; Brundage et al. lack of COI barcode sequences available in 2011). Therefore, the knowledge of the prima- public databases for some groups, namely ry sarcosaprophagous communities, especially several families of Diptera. in terms of species composition and relative abundance, of a specific area, can be very use- The present study aims to (1) determine spe- ful in medico-legal investigations and cies composition and relative abundance of veterinary surveillance. early carrion colonizers in an urban environ- ment using three different attractants, (2) In order to capture forensically important investigate the effectiveness of the type of trap flies, different types of baits and traps have and bait used in estimating first carrion colo- been developed and tested (Tantawi et al. nizers, comparing the species obtained from 1996; Arnaldos et al. 2001; Oliva 2001; Lec- these experiments with those reported in the cese 2004; Hwang and Turner 2005; Baz et al. literature, and (3) identify species through 2007; d’Almeida and Fraga 2007; DNA barcode region and add new sequence Matuszewski et al. 2010; Brundage et al. records to online databases. 2011). Decomposed tissues, used as baits, constitute cheap substrates with no ethi- Materials and Methods cal implications and have been used in other studies for the collection of early colonizers Study area (Leccese 2004; Hwang and Turner 2005; This study was conducted in Lisbon, Portugal, at the campus of the Faculty of Sciences of the

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Journal of Insect Science: Vol. 14 | Article 77 Farinha et al. University of Lisbon in a small patch of grass Sixty-four collections were made. In each ex- with some ornamental shrubs (38º45’22”N periment, four identical traps were set. Three 9º09’30”W). traps were baited, each with 70 g of pork muscle, pork liver or commercial cat food Trapping, preservation, and identification (fish flavor). A fourth trap, unbaited, was used Because insects are cold blooded (poikilo- as a control. Each trap was set up to catch flies thermic), activity and behavior are highly for 24 hours. After the 24-hour period, traps dependent on daily air temperature (Byrd and were closed and insects were collected for Castner 2010). Given the Portuguese climate identification. The traps were then reset with profile—Mediterranean climate, with warm their respective baits and randomly rotated seasons and cold seasons well defined and in- through four positions corresponding to the terspersed with mild seasons—field work cardinal points. Thus, by the end of each ex- experiments were carried out in two groups: periment, each bait had been used once at the first one with an average daily air tem- every of the four positions, so that any site perature above 16ºC (high temperature group; bias should not affect treatment comparisons. 29th March – 1st April 2011 and 3rd – 6th Au- All baits containing eggs were transported in- gust 2011), and a second one with an average to the lab and reared to adults in order to daily temperature below 16ºC (low tempera- obtain the species identification. Air tempera- ture group; 13th – 16th December 2011 and 6th ture was daily recorded using data loggers. – 9th January 2012). This threshold was de- Strong winds and heavy rain prevent flies termined based on the meteorological data of from dispersing (Digby 1958). Therefore, daily temperatures in Lisbon in the previous traps were only set when favorable weather five years. It corresponds to the average daily was present. Collected flies were either pre- air temperature of the months with mild tem- served in 70% ethanol or dried. Insects were peratures (official data from The Geophysical morphologically identified using identification Institute D. Luís, http://idl.ul.pt/). The mean keys (Rognes 1991; Gregor et al. 2002; atmospheric temperature measured during the Oosterbroek 2006) and by specialists whenev- four experiments is presented in Table 1. er required. Only specimens from Calliphoridae, Muscidae, Fanniidae, An- To collect sarcosaprophagous insects, a modi- thomyiidae, and Heleomyzidae families were fied version of the bottle trap was used identified to species level. All specimens were (Hwang and Turner 2005). Traps consisted of deposited in the collection of the Department an empty water carboy with a small frontal of Animal Biology in the Faculty of Sciences opening for the entry of the flies. A grid plat- of the University of Lisbon. form was set over the opening to support the bait. The bottom of the carboy was filled with Statistical analysis water and some drops of detergent to lower Absolute and relative specimen abundance the surface tension of the water and catch the and species richness were analyzed. Families flies that fell down. A transparent plastic tube and genera not identified to species level were with a glass vial at the end was inserted into counted as one species. To estimate the simi- the bottleneck in order to catch adult speci- larity of the communities captured among mens that flew upwards. baits, Sørensen’s similarity coefficient (CS) was used. Its formula is: 2jN / (aN+bN), where aN is the total number of taxa in site A,

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Journal of Insect Science: Vol. 14 | Article 77 Farinha et al. bN is the total number of taxa in site B, and tained 1x colorless GoTaq® Reaction Buffer jN is the sum of the taxa found in both sites. (Promega, www.promega.com), 2 mM MgCl2, This index is equal to 1 in case of total simi- 0.1 mM dNTPs, 0.4 µM of each primer, 0.16 larity and equal to zero if the sites are µg/µL of BSA, 0.25 U of GoTaq® DNA pol- dissimilar and have no species in common. ymerase (Promega), and 4 µL of DNA template, in a final volume of 25 µL. PCR One-way ANOVA was used to test the differ- thermal conditions consisted of 1 cycle of 1 ences among the three baits total captures. A min at 94 °C; 5 cycles of 30 sec at 94 °C, 1 multivariate ANOVA was performed on aver- min at 45 °C, and 1 min at 72 °C;, 35 cycles age values of collected specimens to analyze of 1 min at 94 °C, 1.5 min at 50 °C, and 1 min the effect of the two factors: type of bait and at 72°C; and a final cycle of 5 min at 72°C. temperature. Separate ANOVA analyses were PCR products were verified in 1% agarose done within each forensically important spe- gels stained with RedSafeTM (iNtRON Bio- cies to test the effect of bait in the number of technology, www.intronbio.com) at 100V for collected specimens, and a Mann-Whitney- 25 minutes along with the molecular weight Wilcoxon test was used to analyze the popula- marker HyperLadderTM IV (Bioline, tions between the two temperature groups. www.bioline.com) and then purified using the The Tukey and Bonferroni tests were used to commercial kit SureClean (Bioline) according analyze all post-hoc pair-wise comparison. to the manufacturer’s instructions. Amplicons Data were log transformed in order to get par- were sequenced in both directions by the ametric tests presumptions. Differences were commercial facility offered by Macrogen considered to be significant at the 0.05 level. Company (Macrogen Europe, Analyses were performed using IBM SPSS www.macrogen.com). Statistics 19 (www.ibm.com). DNA sequence analysis DNA extraction, amplification, and se- Sequences were checked and edited in Se- quencing quencher® v4.0.5 (Gene Codes Corporation, A specimen of each Diptera identified to the www.genecodes.com) and then they were species level was subject to molecular identi- blasted in online databases fication using the COI barcode region. (www.ncbi.nlm.nih.gov and www.boldsystems.org). Sequences of all taxa DNA was extracted from two or three legs of identified to species level were submitted to each specimen using the E.Z.N.A.® Insect the Barcode of Life Data Systems (BOLD DNA Kit (Omega Bio-Tek, Systems) database. www.omegabiotek.com), following the manu- facturer’s instructions with the exception of Results the elution step, which was performed in 40 µL of elution buffer in order to increase DNA A total of 2371 specimens belonging to three concentration. COI barcode region (658 bp) insect orders (Diptera, Coleoptera, and Hyme- was amplified using the primer pair LCO1490 noptera) were caught in four experiments (GGTCAACAAATCATAAAGATATTGG) between March 2011 and January 2012 (Table and HCO2198 2). Orders Coleoptera and Hymenoptera were (TAAACTTCAGGGTGACCAAAAAATCA) not analyzed. Diptera was the predominant (Folmer et al. 1994). Each PCR reaction con- order, with a total of 2132 adults (90.0% of all

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Journal of Insect Science: Vol. 14 | Article 77 Farinha et al. collected insects) belonging to 21 families. Concerning bait preferences, C. vicina was the Calliphoridae was the dominant family of the dominant species in pork muscle and cat food Diptera community (39.7%), and Muscidae baits, but with no significant preference for was the second (31.7%). Drosophilidae was either of the three baits. L. sericata presented the third most abundant family (21.6%), a statistically significant preference (P < 0.05) Fanniidae the fourth (7.1%), Sarcophagidae to pork liver bait. and Phoridae were the fifth and sixth, with the same abundance (6.3% each), and Anthomyi- Chrysomya albiceps (Wiedemann), Hydrotaea idae was the seventh (5.1%). Fifteen families ignava (Harris), and the genera Fannia Robi- (mainly non-Calyptrate) were uncommon in neau-Desvoidy and Sarcophaga Meigen the traps (< 2%) (Table 2). presented no significant preference for any of the baits, although all had very low capture Bait variability numbers in cat food bait. From the 2371 collected specimens, 1146 were gathered in pork muscle traps (48.3 %), Concerning the variable temperature, signifi- 834 in pork liver traps (35.2 %) and 391 in cat cant differences in mean captures among the food traps (16.5 %). An ANOVA test revealed three baits in each temperature group were that there are statistical differences between found (high temperature: F = 4.440, P < 0.01; the three baits captures (F = 8.597, P < 0.01). low temperature: F = 3.680, P < 0.05, respec- Posteriori Tukey and Bonferroni comparisons tively), as well as in each bait between the two showed that pork muscle was significantly temperatures (P < 0.01), as warmer tempera- different from both pork liver (P < 0.05) and tures attracted more insects, representing cat food (P < 0.01), the last two being not sig- 79.0% of the total number of specimens cap- nificantly different from each other. tured (Figure 1).

Regarding species richness, pork muscle was In terms of species richness, warmer tempera- the bait with the highest number of species tures provided higher values (Table 2). Taxa trapped, followed by pork liver bait and final- similarity of captured populations for each ly cat food (Table 2). However, when the bait between the two temperature groups re- collected populations of each bait were ana- vealed a very low Sørensen’s similarity lyzed two by two using the Sørensen’s coefficient, being below 0.40 for all baits (Ta- similarity coefficient, few differences were ble 4). found. All comparisons presented CS values ranging between 0.74 (for pork muscle and cat Of the forensically important flies collected, food comparison) and 0.77 (for pork muscle C. vicina was the dominant species in average and pork liver). The comparison between pork low temperatures. L. sericata, L. caesar, L. liver and cat food presented a CS of 0.76 (Ta- ampullacea, C. albiceps, H. ignava, M. do- ble 3). mestica, and the genera Fannia and Sarcophaga, all showed high numbers of cap- Calliphora vicina Robineau-Desvoidy was the tures when average air temperature was above most abundant species (22.0% of all Diptera 16ºC. Moreover, except for Fannia sp., none captured), followed by Musca domestica L., of these species was even collected in the ex- and Lucilia sericata (Meigen) (Table 2). periments with average temperatures below 16ºC. All these taxa showed significant abun-

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Journal of Insect Science: Vol. 14 | Article 77 Farinha et al. dance differences between the high and low Lucilia caesar (L.), Lucilia sericata, average temperature groups (P < 0.01). Graphomyia maculata (Scopoli), Musca do- mestica (L.), levida (Harris), Regarding oviposition in warmer tempera- Muscina stabulans (Fallen), Potamia littoralis tures, it occurred in all three baits, with both (Robineau-Desvoidy), Synthesiomyia nudiseta L. sericata and Sarcophaga sp. laying eggs. In (van der Wulp), Fannia canicularis (L.), An- the lower temperature experiments, oviposi- thomyia pluvialis (L.), Delia platura tion was only observed in two baits, pork (Meigen). Nine species were identified at the muscle and pork liver, and C. vicina was the genus level, Melinda viridicyanea (Robineau- only species that oviposited. Desvoidy), Pollenia rudis (Fabricius), Hydro- taea armipes (Fallen), Muscina pascuorum When both bait and temperature were ana- (Meigen), Muscina prolapsa (Harris), Fannia lyzed together, no interaction was found aequilineata (Ringdahl), Fannia monilis (Hal- between the two variables, i.e., the effects of iday), Anthomyia imbrida (Rondani), and both variables are independent (F = 0.038; P Pegomya ulmaria (Rondani), six to the family > 0.05). level, nigra (Robineau- Desvoidy), Hydrotaea capensis (Wiedemann), Small bait trap efficiency: comparison with Hydrotaea ignava, Limnophora obsignata other studies (Rondani), Fannia pusio (Wiedemann), and The results obtained in the present study re- Lasiomma seminitidum (Zetterstedt), and four garding the presence of forensically important only to order level, Phaonia trimaculata Calliphoridae and Muscidae species were (Bouché), Suillia flagripes (Czerny), Suillia compared with those reported in literature variegata (Loew), and Tephrochlamys ru- from the Iberian Peninsula and are discussed fiventris (Meigen). below in the Discussion section (Tables 5, 6). Discussion Molecular identification through COI bar- code region Species composition and relative abun- From each one of the 34 species identified dance based on morphologic criteria (Table 2), a As in former forensic studies carried out in the specimen was identified at the molecular level Iberian Peninsula, the community of sampled and each COI barcode sequence was submit- Diptera was dominated by the families Calli- ted to BOLD Systems online database. A phoridae and Muscidae (Martínez-Sánchez et successful identification is achieved when the al. 2000; Arnaldos et al. 2004; Baz et al. 2007; nucleotide sequence of the specimen matches Prado e Castro et al. 2012). However, in con- to the available reference sequences, leading trast with these authors, this study presents to a species match. Nevertheless, online data- Drosophilidae as the third most abundant fam- bases are still growing and many species do ily. This can be due to the type of bait or the not have a reference sequence to be compared type of trap used (totally covered in compact with. Of the 34 species morphologically iden- plastic instead of wire mesh), which did not tified, only 15 were correctly identified at the allow any fly, even the smallest, to escape. species level in BOLD database: Calliphora Despite having already recovered some larvae vicina, Calliphora vomitoria (L.), Chrysomia of this family in human autopsies performed albiceps, Lucilia ampullacea (Villeneuve), in Madrid, its forensic importance is still lim-

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Journal of Insect Science: Vol. 14 | Article 77 Farinha et al. ited due to lack of knowledge of its biology 2012; Prado e Castro et al. 2012; George et al. and distribution in the Iberian Peninsula (Ve- 2013). The highest number of specimens and lásquez et al. 2010). Thus, it was not species was caught with a high average air evaluated in the present study. temperature, while the lowest values were ob- tained in low temperatures, which is in Bait variability agreement with the results obtained from Analyzing the factor bait, it was the pork western Spain by Martínez-Sánchez et al. muscle that captured the highest number of (2000). However, Prado e Castro et al. (2012), specimens and species. This result might be in a study conducted in Lisbon, as was the due to the fact that this bait had an outer layer present one, using pig carcasses as bait, pre- that was not as dry as the pork liver and cat sented higher values of specimen abundance food during the experiments, which kept the in spring, a mild season. This inconsistency odor stronger (personal observation). This fea- may be due to differences in size and type of ture might also be the explanation for its large bait, differences in specific biotopes, or tem- masses of eggs, as ecological studies carried perature differences between sampling years, out by Barton-Browne (1962) showed that as Prado e Castro conducted the study in female carrion breeding sites depend on tarsal 2006, when spring had an average temperature contact with water or moisture. In fact, some of 18.9ºC, which was abnormally above the studies aiming to estimate primary forensic mild seasons average. These climate changes colonizers also use pork muscle as bait (e.g., from year to year within seasons highlight the Oliva 2001; Leccese 2004; Baz et al. 2007). In importance of the variable air temperature in- all experiments, the cat food was the least at- stead of calendar limitations (Turchetto and tractive. The absence of eggs in this bait in Vanin 2004). However, it is important to note cold temperatures, when there are fewer active other variables as well, such as the circadian insects, leads to the assumption that it is less cycle and air humidity (Nuorteva 1959; An- preferable to carrion flies and, consequently, derson 2001; Campobasso et al 2001; George not a good substrate to estimate primary Dip- et al. 2013). tera colonizers. Given the abundance of the dominant species When forensically important taxa were ana- at each temperature, we can classify them into lyzed individually, only L. sericata presented groups, the apparent thermophobic (C. vicina) significant differences of abundance among and the apparent thermophilic (L. sericata, L. baits, showing a possible preference for pork caesar, C. albiceps, M. domestica, H. ignava, liver. Fannia sp., and Sarcophaga sp.), all with abundance peaks in the warmer temperatures. Temperature effects on species abundance and richness Small bait traps as an efficient alternative The occurrence of carrion flies was clearly to capture first colonizers: comparison influenced by temperature effects, as expected among studies from the literature (Tantawi et al. 1996; Mar- The overall population of early colonizers col- tínez-Sánchez et al. 2000; Schrӧeder et al. lected in each temperature experiment 2003; Arnaldos et al. 2004; Grassberger and regarding pork muscle bait, because of its Frank 2004; Hwang and Turner 2005; Anton higher species richness and abundance results, et al. 2011; Battán Horenstein and Salvo was compared with populations obtained, for

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Journal of Insect Science: Vol. 14 | Article 77 Farinha et al. the first seven days, in forensic studies using same species of muscid flies as the ones found carcasses conducted in the Iberian in this study, except for the species H. denti- Peninsula (Tables 5, 6). The aim was to de- pes was collected in Prado e Castro et al. 2012 termine whether small bait traps are a viable but not here, and a trade in the genera alternative to other traps with ethical and prac- Phaonia, as the present study collected the ticability constraints. species Phaonia trimaculata and Prado e Cas- tro et al. collected P. subventa (Table 6). Records from studies conducted in Salaman- These results suggest that forensically im- ca, Spain (Martínez-Sánchez et al. 2000), less portant Muscidae communities are more than 500 km away from our sampling sites, region-specific and/or more bait sensitive than showed a similar taxonomic composition in Calliphoridae. All species but Lucilia illustris regard to blowfly species, with all but one documented in human autopsies in northern species (L. ampullacea) collected here not be- Portugal (Cainé et al. 2009) were also collect- ing recovered at Salamanca study sites (Table ed in this study. Nevertheless, caution should 5). A study carried out in Lisbon (Prado e be taken respecting the difficulty in the mor- Castro et al. 2012) presented some differences phologic and molecular differentiation of L. with the present one in the high temperature, caesar and L. illustris, which can lead to mis- namely, the dominance of L. caesar instead of identifications (Oliveira et al. 2011; Sonet et L. sericata concerning family Calliphoridae al. 2012). and the dominance of H. ignava in summer in place of M. domestica regarding family Mus- This analysis suggests that bottle traps with cidae. These differences are consistent with pork muscle as bait are able to capture im- the rural environment in the sampling area of portant early colonizers of carrion and thus Prado e Castro et al. (2012) and the more ur- can be accurate predictors of the Diptera pri- ban environment of the present study mary carrion community, in terms of species (Anderson and Vanlaerhoven 1996; Schrӧeder presence, in a given area. In fact, the traps et al 2003; Hwang and Turner 2005; Brundage used in this study, as well as the experimental et al 2011). Nevertheless, all blowfly species, design, captured the same diversity of Diptera except for Chrysomya megacephala, which early colonizers when compared with studies was collected on pig carcass in Lisbon (Prado using pig carcasses in the same region (Prado e Castro et al. 2012) and also on chicken car- e Castro et al. 2012). Moreover, this kind of cass in Murcia, Southeast Spain (Arnaldos et trap is cheap, easy to make and replicate, and al. 2004), were also collected in the present does not have the ethical, legal, or practicabil- study. It is important to highlight that C. meg- ity implications associated with the use of acephala is a species only recently discovered human remains or pig carcasses. Gruner et al. in Portugal and in low numbers (Prado e Cas- (2007) had already found that aerial capture of tro and García 2009). Conversely, most of the adult specimens accurately predicted carrion- Diptera species from family Muscidae docu- colonizing species. With respect to bait, other mented on the first seven days in carcasses in authors using meat baits or small carcasses Salamanca (Martínez-Sánchez et al. 2000) have already shown that these substrates are were not part of the insect fauna of our study. suitable for the development cycles (Leccese Although, the study performed in Murcia (Ar- 2004) and population estimation of the early naldos et al. 2004) and the one performed in necrophagous Diptera species (Leccese 2004; Lisbon (Prado e Castro et al. 2012) found the Arnaldos et al. 2005; Brundage et al. 2011)

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Journal of Insect Science: Vol. 14 | Article 77 Farinha et al. even though the size of these baits is not com- Conclusions parable to a human body. In fact, the odors This study was the first one carried out in Por- emitted from meat baits should contain many tugal comparing small-bait efficiency in of the volatile organic compounds emitted estimating populations of first colonizers in a from an entire freshly dead mammal, such as specific region. Molecular identification of pigs, the most common bait used in forensic several Diptera species through COI barcode studies (George et al 2013). region, including several species in which such approach was performed for the first Molecular identification time, contributes to increase online databases, In this study, identification of forensically im- thereby expanding the reliability and unequiv- portant Diptera was conducted both at a ocal species determination, which is essential morphological and molecular level. Molecular in many scientific fields, namely in general identification has become an indispensable entomology and in medical-legal investiga- tool in forensic scenarios. The DNA barcod- tions. ing project (www.barcodeoflife.org) aims to assign every species with a unique sequence Although these results have limited applicabil- (barcode sequence), allowing a reliable and ity in criminal investigations of bodies error free identification of a specimen. How- discovered after a long period of time (more ever, this method has its constraints. Closely than one week), the method described here related species may exhibit a very similar nu- proved to be efficient and prompt in estimat- cleotide sequence, and this can lead to a ing the population of early Diptera colonizers. misleading species determination. Examples Therefore, it is a reliable tool to acquire useful of this are L. caesar and L. illustris, which are knowledge to be applied in investigations on not distinguished through COI barcode region fresh body findings, as it easily records the (Wells et al. 2007; Oliveira et al. 2011). Also, primary Diptera sarcosaprophagous fauna of the lack of reference sequences for a great the area where the body was discovered. The- number of species, mainly due to a poor se inventories can help criminal investigations knowledge of species catalogue in some re- in the identification of the species collected gions, leads to identifications only to genus, from the cadaver, human or animal, in indicat- family, or even order level. This last con- ing correct times of oviposition, and also straint was present in this study, as less than signaling the original location of a crime. 50% of the specimens, previously identified morphologically, were blasted in BOLD Sys- Acknowledgements tems database at species level. Few molecular studies on forensically important insects using We are grateful to Verner Michelsen (Univer- COI barcode region have been done in Portu- sity of Copenhagen, Denmark), Andrzej J. gal, except for the most abundant forensic Woźnica (Agricultural University of Diptera (Cainé et al. 2006, 2009; Ferreira et Wroclaw, Poland), Andrzej Grzywacz, and al. 2011; Oliveira et al. 2011; Rolo et al. Krzysztof Szpila (Nicolaus Copernicus Uni- 2013). All sequences were added to the versity in Torun, Poland) for their help in BOLD database and are an important contri- specimen identification. We thank Sara Nar- bution to future species determination through ciso and Marília Antunes (Faculty of molecular methodologies. Sciences, University of Lisbon, Portugal) for assistance in data analyses. We would like to

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Journal of Insect Science: Vol. 14 | Article 77 Farinha et al. thank Computational Biology and Population evidence. Forensic Science International 149: Genomics Group (CoBiG2) for bioinformatics 57-65. support. This work was financially supported by Fundação para a Ciência e Tecnologia, Barton-Browne L. 1962. The relationship Portugal, through the grant PTDC/SAU- between oviposition in the blowfly Lucilia ESA/101228/2008. cuprina and the presence of water. Journal of Insect Physiology 8: 383-390.

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Eberhardt TL, Elliot DA. 2008. A preliminary Battán Horenstein M, Salvo A. 2012. investigation of insect colonization and Community dynamics of carrion flies and succession on remains in New Zealand. their parasitoids in experimental carcasses in Forensic Science International 176: 217-223. Central Argentina. Journal of Insect Science 12(8). Available online: Ferreira S, Oliveira AR, Farinha A, Rebelo www.insectscience.org/12.8 MT, Dias D. 2011. Forensic entomology: Nuclear and mitochondrial markers for Hwang C, Turner BD. 2005. Spatial and Diptera and Coleoptera identification. temporal variability of necrophagous Diptera Forensic Science International: Genetics from urban to rural areas. Medical and Supplement Series 3: e174-e175. Veterinary Entomology 19: 379-391.

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Journal of Insect Science: Vol. 14 | Article 77 Farinha et al.

Figure 1. Distribution of collected specimens by bait in each temperature group. High quality figures are available online.

Table 1. Temperature data (daily average) recorded during the experimental periods (Min - minimum, Max - maximum, Avg - average, SD - standard deviation).

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Journal of Insect Science: Vol. 14 | Article 77 Farinha et al.

Table 2. Insects collected in Lisbon, Portugal, between March 2011 and January 2012, sorted by abundance and bait attraction in each season.

*Oviposition; HT: high temperatures; LT: low temperatures.

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Journal of Insect Science: Vol. 14 | Article 77 Farinha et al. Table 3. Similarity between different seasons and in each bait, expressed by the quantitative Sørensen similarity coefficient (CS).

Table 4. Similarity between the different baits, expressed by the quantitative Sørensen similarity coefficient (CS).

Table 5. Species checklist of Calliphoridae flies from four Iberian studies and those collected in the current study.

*Oviposition; HT: high temperatures; LT: low temperatures. Shaded squares represent the predominant species.

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Journal of Insect Science: Vol. 14 | Article 77 Farinha et al.

Table 6. Species checklist of Muscidae and families Fanniidae and Sarcophagidae flies from four Iberian studies and those collected in the current study.

*Oviposition; HT: high temperatures; LT: low temperatures. Shaded squares represent the predominant species.

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