insects Article Connecting the Dots: From an Easy Method to Computerized Species Determination Senta Niederegger 1,*, Klaus-Peter Döge 2, Marcus Peter 2, Tobias Eickhölter 2 and Gita Mall 1 1 Institute of Legal Medicine, University Hospital Jena, Am Klinikum 1, 07747 Jena, Germany; [email protected] 2 Department of Electrical Engineering and Information Technology, University of Applied Sciences Jena, Carl-Zeiss-Promenade 2, 07745 Jena, Germany; [email protected] (K.-P.D.); [email protected] (M.P.); [email protected] (T.E.) * Correspondence: [email protected]; Tel.: +49-3641-9397130 Academic Editors: David Rivers and John R. Wallace Received: 8 March 2017; Accepted: 12 May 2017; Published: 18 May 2017 Abstract: Differences in growth rate of forensically important dipteran larvae make species determination an essential requisite for an accurate estimation of time since colonization of the body. Interspecific morphological similarities, however, complicate species determination. Muscle attachment site (MAS) patterns on the inside of the cuticula of fly larvae are species specific and grow proportionally with the animal. The patterns can therefore be used for species identification, as well as age estimation in forensically important dipteran larvae. Additionally, in species where determination has proven to be difficult—even when employing genetic methods—this easy and cheap method can be successfully applied. The method was validated for a number of Calliphoridae, as well as Sarcophagidae; for Piophilidae species, however, the method proved to be inapt. The aim of this article is to assess the utility of the MAS method for applications in forensic entomology. Furthermore, the authors are currently engineering automation for pattern acquisition in order to expand the scope of the method. Automation is also required for the fast and reasonable application of MAS for species determination. Using filters on digital microscope pictures and cross-correlating them within their frequency range allows for a calculation of the correlation coefficients. Such pattern recognition permits an automatic comparison of one larva with a database of MAS reference patterns in order to find the correct, or at least the most likely, species. This facilitates species determination in immature stages of forensically important flies and economizes time investment, as rearing to adult flies will no longer be required. Keywords: forensic entomology; muscle attachment sites; species determination; image processing; correlation; knowledge base 1. Introduction Blowflies often start the biological clock for postmortem interval (PMI) calculation in forensic entomology. They might approach a body within the first hours after death [1–3] for oviposition. The age of the oldest blowfly found on a body or carcass is therefore a good estimate for the minimum postmortem interval or minimum exposure time [4–6]. Laboratory experiments growing larvae of known species under controlled temperature regimes showed that age correlates to the body length of the animals [7–11]. However, even though the larvae of different species look extremely similar, they might grow to different body lengths in the same time and under identical conditions [11]. Species and age determination can therefore be a great challenge when a conglomerate of unidentified larvae is collected from a body in the field. Insects 2017, 8, 52; doi:10.3390/insects8020052 www.mdpi.com/journal/insects Insects 2017, 8, 52 2 of 16 AInsects common 2017, 8, 52 method for species determination is to allow collected larvae to pupate and2 of 16 hatch, and to determine the species by morphological features of the imago [3,12]. This is very time-consuming and requiresA common living method larvae. for Other species methods determination based is on to lightallow microscopycollected larvae rely to onpupate the and morphological hatch, and to determine the species by morphological features of the imago [3,12]. This is very characteristics of the larvae, like the shape of the cephalopharyngeal skeleton (CPS), spiracles, or time-consuming and requires living larvae. Other methods based on light microscopy rely on the spinebandsmorphological [13–22 ].characteristics These methods of the canlarvae, be like hindered the shape by of the the fragmentation cephalopharyngeal or discoloration skeleton (CPS), of the specimenspiracles, by poor or spinebands preservation. [13–22]. Technically These method advanceds can approaches be hindered aim by toward the fragmentation DNA-based or species identificationdiscoloration using of molecularthe specimen biological by poor preservati methodson. [23 Technically–25] or electron advanced microscopy approaches [26 aim,27 ].toward Requiring cost-intensiveDNA-based and species specialized identification equipment, using these molecu methodslar biological might not methods be applicable [23–25] in everyor electron laboratory. microscopy [26,27]. Requiring cost-intensive and specialized equipment, these methods might not be 2. Approachapplicable from in every the Biologicallaboratory. Perspective A2. fastApproach and easyfrom method,the Biological suited Perspective for all larval instars and even applicable to a fragmented or discolored specimen, was developed in 2012: While attempting to locate trichoid sensilla, round A fast and easy method, suited for all larval instars and even applicable to a fragmented or structures were detected on the inside of the cuticula of a number of brachyceran larvae. Further discolored specimen, was developed in 2012: While attempting to locate trichoid sensilla, round investigationstructures revealed were detected that they on the were inside attachment of the cu sitesticula for of transversala number of body brachyceran wall muscles. larvae. Further The thoracic segments,investigation as well asrevealed the first that and they last were abdominal attachment segments, sites for each transversal possess a body unique wall muscle muscles. arrangement, The whilethoracic the remaining segments, abdominal as well as segments the first and share last an abdominal identical patternsegments, [28 each,29]. possess The body a unique wall musculaturemuscle of manyarrangement, larvae is composedwhile the remaining of longitudinal, abdominal diagonal, segmen andts share transversal an identical muscles pattern (Figure [28,29].1 a).The Larval body flies lack circumferentialwall musculature muscles of many as larvae antagonists is composed to the longitudinalof longitudinal, muscles diagonal, [30 and]. The transversal vertical displacementmuscles of the(Figure segments 1a). Larval is therefore flies lack probably circumferential supported muscles by the as antagonists transversal to muscles the longitudinal which aremuscles located [30]. in the externalThe layer, vertical where displacement they form of “blocks” the segments in each is segmenttherefore and probably are directly supported attached by the to thetransversal cuticula [31] muscles which are located in the external layer, where they form “blocks” in each segment and are (Figure1b). directly attached to the cuticula [31] (Figure 1b). Figure 1. (a) Opened larva with complete musculature; (b) The longitudinal muscles were removed Figureto 1.reveal(a) Opened the underlying larva with transversal complete muscle musculature; groups. (b) The longitudinal muscles were removed to reveal the underlying transversal muscle groups. 2.1. Species and Age Determination 2.1. SpeciesThe and muscle Age Determination attachment sites (MAS) of these transversal muscles could easily be visualized by Theremoving muscle the attachmentmuscles fromsites the cu (MAS)ticle and of subsequent these transversal staining with muscles Commassie could brilliant easily blue. be visualized The analysis of MAS revealed that the structures are arranged in rows and form distinct patterns (Figure 2a). by removing the muscles from the cuticle and subsequent staining with Commassie brilliant blue. Ten larvae of each species were prepared in this fashion and the pictures were recorded. A small number The analysis of MAS revealed that the structures are arranged in rows and form distinct patterns of row subsets located at the center of the last two thoracic and the first abdominal segment (Segments 2– (Figure4) 2werea). Ten manually larvae charted. of each Correlating species were rows preparedwere stacked in thisand condensed fashion and into the row pictures patterns were(Figure recorded. 2b) A smalland number described of for row a few subsets forensically located important at the center species of of the blowflies last two (Diptera: thoracic Calliphoridae) and the first Calliphora abdominal segmentvomitoria (Segments, C. vicina 2–4), Lucilia were sericata manually [32], Protophormia charted. terraenovae Correlating [33], and rows Phormia were regina stacked [34]. andThe results condensed into rowconfirmed patterns interspecific (Figure variation2b) and which described allowed for for athe few species forensically differentiation important of these five species species. of There blowflies (Diptera:can be Calliphoridae) variation in theCalliphora number of vomitoria attachment, C. points vicina ,composingLucilia sericata a row[ 32in ],larvaeProtophormia of the same terraenovae age and [33], and Phormia regina [34]. The results confirmed interspecific variation which allowed for the species differentiation of these five species. There can be variation in the number of attachment points Insects 2017, 8, 52