Preliminary assessment of cephalopharyngeal skeleton length and body length of Hemipyrellia ligurriens (Wiedemann) (Diptera: Calliphoridae) larvae as potential parameters to estimate minimum post mortem interval

Preliminary assessment of cephalopharyngeal skeleton length and body length of Hemipyrellia... Entomological evidence can be utilized in forensic investigation to estimate the elapsed time after death or minimum post mortem interval estimation (PMI ). This estimation is based on the age of dipterous specimens feeding on min decomposing human tissues, which commonly refers to their larval body length. The objective of this research was to look for alternatives to larval body length which could be impaired by specimen handling and subsequently causing inaccuracy in PMI estimation. In this research, development of forensic blow fly, Hemipyrellia ligurriens (Wiedemann) min (Diptera: Calliphoridae), based on larval cephalopharyngeal skeleton length, was described for the first time. Development of H. ligurriens was represented by body length and cephalopharyngeal skeleton consisting anterodorsal process to dorsal cornu (ADP-DC), anterodorsal process to ventral cornu (ADP-VC) and dorsal cornu to ventral cornu (DC-VC). The cephalopharyngeal skeletons proportionally developed with larval body development, but periods of plateau existed between 0 and 10 h and 24–30 h, suggesting ecdysis periods of larvae. Data measurements were more consistent in cephalopharyngeal skeleton length than in larval body length, and there were strong positive significant correlations, r >0.85 (p < 0.05). These findings warranted further investigations to examine cephalopharyngeal skeleton as an alternative growth parameter to larval body length. Keywords: Forensic entomology, Morphometric, Blowfly, Development Background LaMotte 1995) and larval stage development (Amendt et In forensic entomology, estimation of minimum post al. 2007). Since there is a gradual increase of larval mortem interval (PMI ) in death investigations is de- length with time, it has been widely employed as a refer- min rived from the assessment of insects or arthropods that ence value to estimate its age and PMI (Byrd and min can be found feeding on decomposing human tissues. Castner 2010). The evaluation of PMI commonly refers to the age of However, there are various factors that can affect lar- min the oldest dipterous larvae collected in forensic case val length in PMI determination (Amendt et al. min based on their growth parameters such as body length, 2011). In forensic practice, it includes the method of kill- width (Day and Wallman 2006), weight (Wells and ing and preserving the larvae that could cause a signifi- cant underestimation in the age values of the larvae (Tantawi and Greenberg 1993; Adams and Hall 2003), * Correspondence: rmzuha@ukm.edu.my UKM Forensics, Faculty of Health Sciences, Basement One Tun Seri Lanang and thus, resulting inaccuracies in PMI calculations. min Library, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia Moreover, handling of preservatives could also alter the Centre for Insect Systematics, Faculty of Science & Technology, Universiti conditions of larval specimens, such as colour changes Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Eliza and Zuha Egyptian Journal of Forensic Sciences (2018) 8:39 Page 2 of 7 and degradation of specimens’ quality (Day and The first blow fly egg batch was transferred into a Wallman 2008; Rosilawati et al. 2014). This also includes rearing container by using feather forceps. Rearing con- storage periods in preservatives that affected larval body tainer consists of 250 ml plastic container with 50 g length and weight (Midgley and Villet 2009; Richards et fresh cow’s liver as food source for the larvae. Liver was al. 2013). placed on 3 cm layer sawdust and separated by a piece To obtain a more reliable growth parameter than lar- of tissue paper. Eggs were reared at room temperature val body length, cephalopharyngeal skeleton length was (23–27 °C, 69–94%RH) overnight. Next day, at 0830 h, proposed as an alternative growth parameter for PMI newly emerged first instar larvae were transferred evenly min estimation (Rabbani and Zuha 2017). By using trad- into five freshly prepared rearing containers. Larval sam- itional morphometric analysis, measurements were ob- pling was conducted twice a day at 0900 h and 1500 h. tained from landmarks distances of cephalopharyngeal During each sampling occasion, three larvae were se- skeletons. This technique is the basis to geometric mor- lected randomly from each container and killed using phometrics which provides ideal tools in shape analysis near-boiling water (≈80 °C) (Adams and Hall 2003). to discriminate insect species or sexual dimorphism (Zelditch et al. 2012; Nuñez-Rodríguez and Liria 2017). Measurement Although there are various applications that can be Larval body length was measured in lateral position found in morphometric analysis regarding shapes and from the tip of the mouth hook to the posterior spiracle growth of biological organisms (Bookstein 1982; Strauss by using Nikon SMZ745T stereomicroscope fitted with and Bookstein 1982; Marcus 1988; James Rohlf and Dino-Lite® camera and Dino Capture 2.0® Software. Marcus 1993), the methodology being used in this We obtained cephalopharyngeal skeletons based on present research was only limited to measuring distances guidelines by Rabbani and Zuha (2017). Segment 1–5of between landmarks (Daly 1985). Furthermore, informa- the larvae were cut and soaked in 10% potassium hy- tion regarding development of calliphorid larvae based droxide (KOH) for 5 min. Dissected segments were care- on cephalopharyngeal skeleton especially in forensic ap- fully washed in 10% KOH to avoid damaging the plication is scarce. cephalopharyngeal skeleton structures, and later In the present study, growth data of Hemipyrellia immersed in 10% acetic acid for 30 s, followed by ligurriens (Wiedemann) (Diptera: Calliphoridae) were immersion in 70% ethanol for 3 min. Then, cephalophar- explained based on its cephalopharyngeal skeleton yngeal skeleton was mounted on a glass slide in lateral length by using a streamline morphometric analysis. It is position by using Euparal, and covered with 7 mm a forensically important blowfly species that predomin- rounded cover slip. Cephalopharyngeal skeleton was antly colonising corpses in Malaysia and Thailand (Lee subsequently measured by using streamline measure- et al. 2004; Bunchu et al. 2012; Kumara et al. 2012). ment landmarks, i.e., anterodorsal process (clipeal arc) Other than charting the cephalopharyngeal skeleton (ADP) to dorsal cornu (DC), ADP to ventral cornu (VC) growth, its correlation with the development of larval and DC to VC (Fig. 1) (Nateeworanart et al. 2010; body length was also determined to show its potential Nuñez and Liria 2016; Rabbani and Zuha 2017). alternative as growth parameter in PMI estimation. min Materials and methods Sample preparation This research was conducted in two study replications throughout period from 14 February 2017 to 22 March 2017. Approximately 300 g decomposed yellowstripe scads (Selaroides leptolepis Cuvier) was used as baits and oviposition medium for adult blow flies. They were placed in a plastic container on the ground and left ex- posed in outdoor environment adjacent to Forensic Entomology Laboratory, Universiti Kebangsaan Malaysia, Bangi (2.92°N, 101.78°E). Baits were left exposed for 3 h to allow oviposition of blow flies and they were frequently checked for any oviposition activity from a single female blow fly. The number of larvae used in this experiment Fig. 1 Developmental landmarks of H. ligurriens based on cephalopharyngeal skeleton length (ADP-DC Anterodorsal was based on the total number of eggs obtained from a process-dorsal cornua, ADP-VC: Anterodorsal process-ventral single female blow fly which consists of 87 (study replica- cornua, DC-VC Dorsal cornua-ventral cornua) (Bar = 0.5 mm) tion 1) and 71 eggs (study replication 2), respectively. Eliza and Zuha Egyptian Journal of Forensic Sciences (2018) 8:39 Page 3 of 7 Sampling and measurement protocols were repeated number of slits in the posterior spiracles (de Carvalho in the next sampling and stopped when the larvae Queiroz et al. 1997; Thyssen and Linhares 2007). reached post feeding stage. Descriptive analysis was car- In this present study, larval body length and cephalo- ried out to obtain the mean value of the length and pharyngeal skeleton length of H. ligurriens were further within-group sample variance while Spearman’s rho cor- explored by using traditional morphometrics and relation test was used to determine correlation between descriptive analysis (Table 1). Consistencies of measure- body length and cephalopharyngeal skeleton (ADP-DC, ment values based on body length and cephalopharny- ADP-VC, DC-VC). The statistical tests were performed geal skeleton length were represented by within-group by using SPSS Ver. 22.0. sample variance (s ). In both study replications, vari- ances of ADP-DC, ADP-VC and DC-VC were smaller Species identification than variances of body length, indicating that measure- Species identifications were conducted on the third in- ments of individual values in cephalopharyngeal skeleton star larvae of the sample and adults that emerged from were more consistent than body length. This proved the the remaining larvae colony based on local Calliphoridae underlying issues when using larval body length as identification keys (Kurahashi et al. 1997; Greenberg and growth parameter in PMI estimation, that the scler- min Kunich 2002). It was found that all specimens used in ites of cephalopharyngeal skeleton were more rigid than this study were H. ligurriens. larval soft bodies which vulnerable to physical distor- tions. Therefore, in this study, ADP-DC, ADP-VC and Results and discussion DC-VC were chosen as the suitable landmarks to repre- The development from first instar until peak feeding third sent the cephalopharyngeal skeleton. Previous study in- instar larvae of H. ligurriens took approximately 54 h in cluded mouth hooks of the cephalopharyngeal skeleton both replications (Fig. 2). Growth patterns based on mean as landmark for measurements, but there were possible lengths were observed more gradually increased in body drawbacks because they were easily affected by move- lengths compared to mean cephalopharyngeal skeleton ment during cleaning process, especially in the first and lengths. Development of cephalopharyngeal skeletons also second instar larvae (Rabbani and Zuha 2017). As ob- exhibited periods of plateau between 0 and 10 h and served in other calliphorids such as Chrysomya albiceps 24–30 h (ADP-DC & ADP-VC) in study replication 1. In (Wiedemann) (de Carvalho Queiroz 1997) and Hypopy- study replication 2, similar patterns were observed in both giopsis fumipennis (Walker) (Heo et al. 2015), different body lengths and cephalopharyngeal skeleton lengths dur- position and shape of mouth hook during different larval ing the first 10 h, but cephalopharyngeal skeleton lengths instars could also affect the length between landmarks. reached their plateau stage at 30 h of development. In subsequent analysis, correlations between body Overall, the growth pattern of cephalopharnygeal skeleton length and cephalopharyngeal skeleton length of larvae was coherent with body larval body length in study repli- were determined to establish their associations. Since cation 1, contrasting the pattern in study replication 2 data were not normally distributed, Spearman’s rho cor- where the growth of cephalopharyngeal skeleton drastic- relation tests were carried out on body length and ally increased from 24 to 30 h. cephalopharyngeal skeleton. Results indicate strong sig- The periods of plateau documented in cephalopharyn- nificant and positive correlations between body lengths geal skeletons were possibly existed because of transi- and cephalopharyngeal skeleton lengths which were rep- tional phase during ecdysis, i.e., from first to second resented by ADP-DC, ADP-VC and DC-VC for H. ligur- instar and from second to third instar. In morphometric riens with correlation coeffiecients, r, ranged 0.85–0.97 studies of larval development, deviations from linear (p < 0.05) (Table 2). The findings suggested that cephalo- growth suggested instar periods for many species includ- pharyngeal skeleton allometry can be used as a growth ing dipterous larvae (Lawrence 1979; Daly 1985). When parameter to describe H. ligurriens development. compared with data from Bunchu et al. (2012), the plea- Allometric growth patterns in dipterous larvae have teau periods of cephalopharyngeal skeleton in study rep- been recorded in few developmental studies by lication 1 could be roughly superimposed on the growth highlighting the measurements of cephalopharyngeal curve of H. ligurriens larval stage. This evaluation, how- skeleton segments, but they were not extended to foren- ever, could be affected by variations of techniques sic application (Lawrence 1979; Petitt 1990). Other than employed in both studies such as rearing temperatures, being useful in species identification and distinguishing sample volumes and larval food types. To overcome this cryptic species (Canal et al. 2015), cephalopharyngeal issue in future research, it is important to conduct a skeleton of dipterous larvae has been used to describe proper documentation during larval ecdysis by associat- the growth patterns (Rabbani and Zuha 2017; Simon et ing the cephalopharyngeal skeleton length with larval in- al. 2011), but the information is still scarce. It is possible, stars based on qualitative assessment such as the from the present findings, allometric growth of Eliza and Zuha Egyptian Journal of Forensic Sciences (2018) 8:39 Page 4 of 7 Fig. 2 Developmental rate of larval body length and cephalopharyngeal skeleton (ADP-DC Anterodorsal process-dorsal cornua, ADP-VC Anterodorsal process-ventral cornua, DC-VC Dorsal cornua-ventral cornua) of H. ligurriens in the first and second study replication Eliza and Zuha Egyptian Journal of Forensic Sciences (2018) 8:39 Page 5 of 7 Table 1 Morphometry of Hemipyrellia ligurriens cephalopharyngeal skeleton (in mm) in the first and second replication based on sampling intervals (h) (BL Body length, ADP-DC Anterodorsal process-dorsal cornua, ADP-VC Anterodorsal process-ventral cornua, DC-VC: Dorsal cornua-ventral cornua) 2 2 Landmarks Sampling M Min Max Range s s NM Min Max Range s s N intervals (h) First Replication Second Replication BL 0 2.769 2.631 3.010 0.379 0.104 0.011 14 3.168 3.004 3.375 0.371 0.114 0.013 11 6 3.192 2.726 3.556 0.830 0.211 0.045 15 3.234 3.026 3.464 0.438 0.132 0.017 9 24 5.785 4.915 6.237 1.322 0.331 0.109 13 6.616 5.978 7.384 1.406 0.394 0.155 12 30 6.534 5.560 6.780 1.220 0.351 0.124 15 7.553 6.525 9.912 3.387 0.970 0.940 11 48 11.649 10.348 12.284 1.936 0.473 0.224 15 12.772 11.261 13.990 2.729 0.738 0.545 14 54 12.393 11.712 13.081 1.369 0.350 0.122 15 13.586 12.901 14.642 1.741 0.560 0.314 14 ADP-DC 0 0.128 0.107 0.141 0.034 0.010 0.000 14 0.141 0.134 0.148 0.014 0.004 0.000 11 6 0.134 0.119 0.151 0.032 0.008 0.000 15 0.134 0.114 0.155 0.041 0.014 0.000 9 24 0.462 0.419 0.479 0.060 0.017 0.000 13 0.432 0.359 0.473 0.114 0.033 0.001 12 30 0.455 0.426 0.490 0.064 0.020 0.000 15 0.967 0.816 1.069 0.253 0.066 0.004 11 48 0.999 0.919 1.046 0.127 0.044 0.002 15 0.954 0.864 1.059 0.195 0.057 0.003 14 54 1.025 0.992 1.071 0.079 0.028 0.001 15 0.976 0.910 1.072 0.162 0.048 0.002 14 ADP-VC 0 0.151 0.128 0.174 0.046 0.013 0.000 14 0.149 0.127 0.174 0.047 0.011 0.000 11 6 0.149 0.109 0.174 0.065 0.017 0.000 15 0.142 0.131 0.162 0.031 0.010 0.000 9 24 0.373 0.344 0.392 0.048 0.016 0.000 13 0.382 0.342 0.408 0.066 0.017 0.000 12 30 0.360 0.240 0.402 0.162 0.039 0.002 15 0.730 0.642 0.832 0.190 0.048 0.002 11 48 0.750 0.669 0.814 0.145 0.044 0.002 15 0.734 0.662 0.819 0.157 0.043 0.002 14 54 0.785 0.716 0.835 0.119 0.033 0.001 15 0.743 0.680 0.800 0.120 0.032 0.001 14 DC-VC 0 0.104 0.075 0.137 0.062 0.017 0.000 14 0.098 0.079 0.114 0.035 0.013 0.000 11 6 0.097 0.056 0.124 0.068 0.016 0.000 15 0.094 0.081 0.114 0.033 0.010 0.000 9 24 0.168 0.134 0.198 0.064 0.022 0.000 13 0.192 0.129 0.225 0.096 0.029 0.001 12 30 0.188 0.146 0.390 0.244 0.059 0.003 15 0.322 0.256 0.371 0.115 0.040 0.002 11 48 0.359 0.310 0.441 0.131 0.041 0.002 15 0.335 0.253 0.425 0.172 0.050 0.003 14 54 0.378 0.326 0.420 0.094 0.026 0.001 15 0.349 0.283 0.385 0.102 0.032 0.001 14 Eliza and Zuha Egyptian Journal of Forensic Sciences (2018) 8:39 Page 6 of 7 Table 2 Non-parametric correlation analysis using Spearman’s Equipment used is this research was partially funded by the second author’s Young Researcher’s Incentive Scheme (Geran Galakan Penyelidik Muda) rho (r) correlation coefficient and p value of relationships GGPM-2014-018. between cephalopharyngeal skeleton and body length of H. ligurriens in the first and second replication Availability of data and materials Replication Growth Parameters BL ADP-DC ADP-VC DC-VC Data is available by request to the corresponding author: rmzuha@ukm.edu.my. 1st BL r = 0.921 r = 0.914 r = 0.894 Authors’ contributions p < 0.05 p < 0.05 p < 0.05 PE and RMZ conducted the research and organised the structure of the manuscript. Both authors read and approved the final manuscript. ADP-DC r = 0.921 r = 0.961 r = 0.904 p < 0.05 p < 0.05 p < 0.05 Authors’ information PE was an undergraduate student and supervised by RMZ, the project leader ADP-VC r = 0.914 r = 0.961 r = 0.901 and a forensic entomology consultant at Universiti Kebangsaan Malaysia p < 0.05 p < 0.05 p < 0.05 (UKM). RMZ is currently attached to UKM Forensics and a researcher at Center for Insect Systematics, UKM. DC-VC r = 0.894 r = 0.904 r = 0.901 p < 0.05 p < 0.05 p < 0.05 Ethics approval and consent to participate Not applicable. 2nd BL r = 0.853 r = 0.852 r = 0.855 p < 0.05 p < 0.05 p < 0.05 Competing interests The authors declare that they have no competing interests. ADP-DC r = 0.853 r = 0.968 r = 0.909 p < 0.05 p < 0.05 p < 0.05 Publisher’sNote ADP-VC r = 0.852 r = 0.968 r = 0.898 Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. p < 0.05 p < 0.05 p < 0.05 DC-VC r = 0.855 r = 0.909 r = 0.898 Received: 28 November 2017 Accepted: 1 June 2018 p < 0.05 p < 0.05 p < 0.05 References Adams ZJO, Hall MJR (2003) Methods used for the killing and preservation of forensically important larvae could be explored further blowfly larvae, and their effect on post-mortem larval length. Forensic Sci Int on its patterns and developmental model. 138:50–61. https://doi.org/10.1016/j.forsciint.2003.08.010. Amendt J, Campobasso CP, Gaudry E, Reiter C, LeBlanc HN, Hall MJR (2007) Best practice in forensic entomology—standards and guidelines. Int J Legal Med Conclusions 121:90–104. 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Preliminary assessment of cephalopharyngeal skeleton length and body length of Hemipyrellia ligurriens (Wiedemann) (Diptera: Calliphoridae) larvae as potential parameters to estimate minimum post mortem interval

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Abstract

Entomological evidence can be utilized in forensic investigation to estimate the elapsed time after death or minimum post mortem interval estimation (PMI ). This estimation is based on the age of dipterous specimens feeding on min decomposing human tissues, which commonly refers to their larval body length. The objective of this research was to look for alternatives to larval body length which could be impaired by specimen handling and subsequently causing inaccuracy in PMI estimation. In this research, development of forensic blow fly, Hemipyrellia ligurriens (Wiedemann) min (Diptera: Calliphoridae), based on larval cephalopharyngeal skeleton length, was described for the first time. Development of H. ligurriens was represented by body length and cephalopharyngeal skeleton consisting anterodorsal process to dorsal cornu (ADP-DC), anterodorsal process to ventral cornu (ADP-VC) and dorsal cornu to ventral cornu (DC-VC). The cephalopharyngeal skeletons proportionally developed with larval body development, but periods of plateau existed between 0 and 10 h and 24–30 h, suggesting ecdysis periods of larvae. Data measurements were more consistent in cephalopharyngeal skeleton length than in larval body length, and there were strong positive significant correlations, r >0.85 (p < 0.05). These findings warranted further investigations to examine cephalopharyngeal skeleton as an alternative growth parameter to larval body length. Keywords: Forensic entomology, Morphometric, Blowfly, Development Background LaMotte 1995) and larval stage development (Amendt et In forensic entomology, estimation of minimum post al. 2007). Since there is a gradual increase of larval mortem interval (PMI ) in death investigations is de- length with time, it has been widely employed as a refer- min rived from the assessment of insects or arthropods that ence value to estimate its age and PMI (Byrd and min can be found feeding on decomposing human tissues. Castner 2010). The evaluation of PMI commonly refers to the age of However, there are various factors that can affect lar- min the oldest dipterous larvae collected in forensic case val length in PMI determination (Amendt et al. min based on their growth parameters such as body length, 2011). In forensic practice, it includes the method of kill- width (Day and Wallman 2006), weight (Wells and ing and preserving the larvae that could cause a signifi- cant underestimation in the age values of the larvae (Tantawi and Greenberg 1993; Adams and Hall 2003), * Correspondence: rmzuha@ukm.edu.my UKM Forensics, Faculty of Health Sciences, Basement One Tun Seri Lanang and thus, resulting inaccuracies in PMI calculations. min Library, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia Moreover, handling of preservatives could also alter the Centre for Insect Systematics, Faculty of Science & Technology, Universiti conditions of larval specimens, such as colour changes Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Eliza and Zuha Egyptian Journal of Forensic Sciences (2018) 8:39 Page 2 of 7 and degradation of specimens’ quality (Day and The first blow fly egg batch was transferred into a Wallman 2008; Rosilawati et al. 2014). This also includes rearing container by using feather forceps. Rearing con- storage periods in preservatives that affected larval body tainer consists of 250 ml plastic container with 50 g length and weight (Midgley and Villet 2009; Richards et fresh cow’s liver as food source for the larvae. Liver was al. 2013). placed on 3 cm layer sawdust and separated by a piece To obtain a more reliable growth parameter than lar- of tissue paper. Eggs were reared at room temperature val body length, cephalopharyngeal skeleton length was (23–27 °C, 69–94%RH) overnight. Next day, at 0830 h, proposed as an alternative growth parameter for PMI newly emerged first instar larvae were transferred evenly min estimation (Rabbani and Zuha 2017). By using trad- into five freshly prepared rearing containers. Larval sam- itional morphometric analysis, measurements were ob- pling was conducted twice a day at 0900 h and 1500 h. tained from landmarks distances of cephalopharyngeal During each sampling occasion, three larvae were se- skeletons. This technique is the basis to geometric mor- lected randomly from each container and killed using phometrics which provides ideal tools in shape analysis near-boiling water (≈80 °C) (Adams and Hall 2003). to discriminate insect species or sexual dimorphism (Zelditch et al. 2012; Nuñez-Rodríguez and Liria 2017). Measurement Although there are various applications that can be Larval body length was measured in lateral position found in morphometric analysis regarding shapes and from the tip of the mouth hook to the posterior spiracle growth of biological organisms (Bookstein 1982; Strauss by using Nikon SMZ745T stereomicroscope fitted with and Bookstein 1982; Marcus 1988; James Rohlf and Dino-Lite® camera and Dino Capture 2.0® Software. Marcus 1993), the methodology being used in this We obtained cephalopharyngeal skeletons based on present research was only limited to measuring distances guidelines by Rabbani and Zuha (2017). Segment 1–5of between landmarks (Daly 1985). Furthermore, informa- the larvae were cut and soaked in 10% potassium hy- tion regarding development of calliphorid larvae based droxide (KOH) for 5 min. Dissected segments were care- on cephalopharyngeal skeleton especially in forensic ap- fully washed in 10% KOH to avoid damaging the plication is scarce. cephalopharyngeal skeleton structures, and later In the present study, growth data of Hemipyrellia immersed in 10% acetic acid for 30 s, followed by ligurriens (Wiedemann) (Diptera: Calliphoridae) were immersion in 70% ethanol for 3 min. Then, cephalophar- explained based on its cephalopharyngeal skeleton yngeal skeleton was mounted on a glass slide in lateral length by using a streamline morphometric analysis. It is position by using Euparal, and covered with 7 mm a forensically important blowfly species that predomin- rounded cover slip. Cephalopharyngeal skeleton was antly colonising corpses in Malaysia and Thailand (Lee subsequently measured by using streamline measure- et al. 2004; Bunchu et al. 2012; Kumara et al. 2012). ment landmarks, i.e., anterodorsal process (clipeal arc) Other than charting the cephalopharyngeal skeleton (ADP) to dorsal cornu (DC), ADP to ventral cornu (VC) growth, its correlation with the development of larval and DC to VC (Fig. 1) (Nateeworanart et al. 2010; body length was also determined to show its potential Nuñez and Liria 2016; Rabbani and Zuha 2017). alternative as growth parameter in PMI estimation. min Materials and methods Sample preparation This research was conducted in two study replications throughout period from 14 February 2017 to 22 March 2017. Approximately 300 g decomposed yellowstripe scads (Selaroides leptolepis Cuvier) was used as baits and oviposition medium for adult blow flies. They were placed in a plastic container on the ground and left ex- posed in outdoor environment adjacent to Forensic Entomology Laboratory, Universiti Kebangsaan Malaysia, Bangi (2.92°N, 101.78°E). Baits were left exposed for 3 h to allow oviposition of blow flies and they were frequently checked for any oviposition activity from a single female blow fly. The number of larvae used in this experiment Fig. 1 Developmental landmarks of H. ligurriens based on cephalopharyngeal skeleton length (ADP-DC Anterodorsal was based on the total number of eggs obtained from a process-dorsal cornua, ADP-VC: Anterodorsal process-ventral single female blow fly which consists of 87 (study replica- cornua, DC-VC Dorsal cornua-ventral cornua) (Bar = 0.5 mm) tion 1) and 71 eggs (study replication 2), respectively. Eliza and Zuha Egyptian Journal of Forensic Sciences (2018) 8:39 Page 3 of 7 Sampling and measurement protocols were repeated number of slits in the posterior spiracles (de Carvalho in the next sampling and stopped when the larvae Queiroz et al. 1997; Thyssen and Linhares 2007). reached post feeding stage. Descriptive analysis was car- In this present study, larval body length and cephalo- ried out to obtain the mean value of the length and pharyngeal skeleton length of H. ligurriens were further within-group sample variance while Spearman’s rho cor- explored by using traditional morphometrics and relation test was used to determine correlation between descriptive analysis (Table 1). Consistencies of measure- body length and cephalopharyngeal skeleton (ADP-DC, ment values based on body length and cephalopharny- ADP-VC, DC-VC). The statistical tests were performed geal skeleton length were represented by within-group by using SPSS Ver. 22.0. sample variance (s ). In both study replications, vari- ances of ADP-DC, ADP-VC and DC-VC were smaller Species identification than variances of body length, indicating that measure- Species identifications were conducted on the third in- ments of individual values in cephalopharyngeal skeleton star larvae of the sample and adults that emerged from were more consistent than body length. This proved the the remaining larvae colony based on local Calliphoridae underlying issues when using larval body length as identification keys (Kurahashi et al. 1997; Greenberg and growth parameter in PMI estimation, that the scler- min Kunich 2002). It was found that all specimens used in ites of cephalopharyngeal skeleton were more rigid than this study were H. ligurriens. larval soft bodies which vulnerable to physical distor- tions. Therefore, in this study, ADP-DC, ADP-VC and Results and discussion DC-VC were chosen as the suitable landmarks to repre- The development from first instar until peak feeding third sent the cephalopharyngeal skeleton. Previous study in- instar larvae of H. ligurriens took approximately 54 h in cluded mouth hooks of the cephalopharyngeal skeleton both replications (Fig. 2). Growth patterns based on mean as landmark for measurements, but there were possible lengths were observed more gradually increased in body drawbacks because they were easily affected by move- lengths compared to mean cephalopharyngeal skeleton ment during cleaning process, especially in the first and lengths. Development of cephalopharyngeal skeletons also second instar larvae (Rabbani and Zuha 2017). As ob- exhibited periods of plateau between 0 and 10 h and served in other calliphorids such as Chrysomya albiceps 24–30 h (ADP-DC & ADP-VC) in study replication 1. In (Wiedemann) (de Carvalho Queiroz 1997) and Hypopy- study replication 2, similar patterns were observed in both giopsis fumipennis (Walker) (Heo et al. 2015), different body lengths and cephalopharyngeal skeleton lengths dur- position and shape of mouth hook during different larval ing the first 10 h, but cephalopharyngeal skeleton lengths instars could also affect the length between landmarks. reached their plateau stage at 30 h of development. In subsequent analysis, correlations between body Overall, the growth pattern of cephalopharnygeal skeleton length and cephalopharyngeal skeleton length of larvae was coherent with body larval body length in study repli- were determined to establish their associations. Since cation 1, contrasting the pattern in study replication 2 data were not normally distributed, Spearman’s rho cor- where the growth of cephalopharyngeal skeleton drastic- relation tests were carried out on body length and ally increased from 24 to 30 h. cephalopharyngeal skeleton. Results indicate strong sig- The periods of plateau documented in cephalopharyn- nificant and positive correlations between body lengths geal skeletons were possibly existed because of transi- and cephalopharyngeal skeleton lengths which were rep- tional phase during ecdysis, i.e., from first to second resented by ADP-DC, ADP-VC and DC-VC for H. ligur- instar and from second to third instar. In morphometric riens with correlation coeffiecients, r, ranged 0.85–0.97 studies of larval development, deviations from linear (p < 0.05) (Table 2). The findings suggested that cephalo- growth suggested instar periods for many species includ- pharyngeal skeleton allometry can be used as a growth ing dipterous larvae (Lawrence 1979; Daly 1985). When parameter to describe H. ligurriens development. compared with data from Bunchu et al. (2012), the plea- Allometric growth patterns in dipterous larvae have teau periods of cephalopharyngeal skeleton in study rep- been recorded in few developmental studies by lication 1 could be roughly superimposed on the growth highlighting the measurements of cephalopharyngeal curve of H. ligurriens larval stage. This evaluation, how- skeleton segments, but they were not extended to foren- ever, could be affected by variations of techniques sic application (Lawrence 1979; Petitt 1990). Other than employed in both studies such as rearing temperatures, being useful in species identification and distinguishing sample volumes and larval food types. To overcome this cryptic species (Canal et al. 2015), cephalopharyngeal issue in future research, it is important to conduct a skeleton of dipterous larvae has been used to describe proper documentation during larval ecdysis by associat- the growth patterns (Rabbani and Zuha 2017; Simon et ing the cephalopharyngeal skeleton length with larval in- al. 2011), but the information is still scarce. It is possible, stars based on qualitative assessment such as the from the present findings, allometric growth of Eliza and Zuha Egyptian Journal of Forensic Sciences (2018) 8:39 Page 4 of 7 Fig. 2 Developmental rate of larval body length and cephalopharyngeal skeleton (ADP-DC Anterodorsal process-dorsal cornua, ADP-VC Anterodorsal process-ventral cornua, DC-VC Dorsal cornua-ventral cornua) of H. ligurriens in the first and second study replication Eliza and Zuha Egyptian Journal of Forensic Sciences (2018) 8:39 Page 5 of 7 Table 1 Morphometry of Hemipyrellia ligurriens cephalopharyngeal skeleton (in mm) in the first and second replication based on sampling intervals (h) (BL Body length, ADP-DC Anterodorsal process-dorsal cornua, ADP-VC Anterodorsal process-ventral cornua, DC-VC: Dorsal cornua-ventral cornua) 2 2 Landmarks Sampling M Min Max Range s s NM Min Max Range s s N intervals (h) First Replication Second Replication BL 0 2.769 2.631 3.010 0.379 0.104 0.011 14 3.168 3.004 3.375 0.371 0.114 0.013 11 6 3.192 2.726 3.556 0.830 0.211 0.045 15 3.234 3.026 3.464 0.438 0.132 0.017 9 24 5.785 4.915 6.237 1.322 0.331 0.109 13 6.616 5.978 7.384 1.406 0.394 0.155 12 30 6.534 5.560 6.780 1.220 0.351 0.124 15 7.553 6.525 9.912 3.387 0.970 0.940 11 48 11.649 10.348 12.284 1.936 0.473 0.224 15 12.772 11.261 13.990 2.729 0.738 0.545 14 54 12.393 11.712 13.081 1.369 0.350 0.122 15 13.586 12.901 14.642 1.741 0.560 0.314 14 ADP-DC 0 0.128 0.107 0.141 0.034 0.010 0.000 14 0.141 0.134 0.148 0.014 0.004 0.000 11 6 0.134 0.119 0.151 0.032 0.008 0.000 15 0.134 0.114 0.155 0.041 0.014 0.000 9 24 0.462 0.419 0.479 0.060 0.017 0.000 13 0.432 0.359 0.473 0.114 0.033 0.001 12 30 0.455 0.426 0.490 0.064 0.020 0.000 15 0.967 0.816 1.069 0.253 0.066 0.004 11 48 0.999 0.919 1.046 0.127 0.044 0.002 15 0.954 0.864 1.059 0.195 0.057 0.003 14 54 1.025 0.992 1.071 0.079 0.028 0.001 15 0.976 0.910 1.072 0.162 0.048 0.002 14 ADP-VC 0 0.151 0.128 0.174 0.046 0.013 0.000 14 0.149 0.127 0.174 0.047 0.011 0.000 11 6 0.149 0.109 0.174 0.065 0.017 0.000 15 0.142 0.131 0.162 0.031 0.010 0.000 9 24 0.373 0.344 0.392 0.048 0.016 0.000 13 0.382 0.342 0.408 0.066 0.017 0.000 12 30 0.360 0.240 0.402 0.162 0.039 0.002 15 0.730 0.642 0.832 0.190 0.048 0.002 11 48 0.750 0.669 0.814 0.145 0.044 0.002 15 0.734 0.662 0.819 0.157 0.043 0.002 14 54 0.785 0.716 0.835 0.119 0.033 0.001 15 0.743 0.680 0.800 0.120 0.032 0.001 14 DC-VC 0 0.104 0.075 0.137 0.062 0.017 0.000 14 0.098 0.079 0.114 0.035 0.013 0.000 11 6 0.097 0.056 0.124 0.068 0.016 0.000 15 0.094 0.081 0.114 0.033 0.010 0.000 9 24 0.168 0.134 0.198 0.064 0.022 0.000 13 0.192 0.129 0.225 0.096 0.029 0.001 12 30 0.188 0.146 0.390 0.244 0.059 0.003 15 0.322 0.256 0.371 0.115 0.040 0.002 11 48 0.359 0.310 0.441 0.131 0.041 0.002 15 0.335 0.253 0.425 0.172 0.050 0.003 14 54 0.378 0.326 0.420 0.094 0.026 0.001 15 0.349 0.283 0.385 0.102 0.032 0.001 14 Eliza and Zuha Egyptian Journal of Forensic Sciences (2018) 8:39 Page 6 of 7 Table 2 Non-parametric correlation analysis using Spearman’s Equipment used is this research was partially funded by the second author’s Young Researcher’s Incentive Scheme (Geran Galakan Penyelidik Muda) rho (r) correlation coefficient and p value of relationships GGPM-2014-018. between cephalopharyngeal skeleton and body length of H. ligurriens in the first and second replication Availability of data and materials Replication Growth Parameters BL ADP-DC ADP-VC DC-VC Data is available by request to the corresponding author: rmzuha@ukm.edu.my. 1st BL r = 0.921 r = 0.914 r = 0.894 Authors’ contributions p < 0.05 p < 0.05 p < 0.05 PE and RMZ conducted the research and organised the structure of the manuscript. Both authors read and approved the final manuscript. ADP-DC r = 0.921 r = 0.961 r = 0.904 p < 0.05 p < 0.05 p < 0.05 Authors’ information PE was an undergraduate student and supervised by RMZ, the project leader ADP-VC r = 0.914 r = 0.961 r = 0.901 and a forensic entomology consultant at Universiti Kebangsaan Malaysia p < 0.05 p < 0.05 p < 0.05 (UKM). RMZ is currently attached to UKM Forensics and a researcher at Center for Insect Systematics, UKM. DC-VC r = 0.894 r = 0.904 r = 0.901 p < 0.05 p < 0.05 p < 0.05 Ethics approval and consent to participate Not applicable. 2nd BL r = 0.853 r = 0.852 r = 0.855 p < 0.05 p < 0.05 p < 0.05 Competing interests The authors declare that they have no competing interests. ADP-DC r = 0.853 r = 0.968 r = 0.909 p < 0.05 p < 0.05 p < 0.05 Publisher’sNote ADP-VC r = 0.852 r = 0.968 r = 0.898 Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. p < 0.05 p < 0.05 p < 0.05 DC-VC r = 0.855 r = 0.909 r = 0.898 Received: 28 November 2017 Accepted: 1 June 2018 p < 0.05 p < 0.05 p < 0.05 References Adams ZJO, Hall MJR (2003) Methods used for the killing and preservation of forensically important larvae could be explored further blowfly larvae, and their effect on post-mortem larval length. Forensic Sci Int on its patterns and developmental model. 138:50–61. https://doi.org/10.1016/j.forsciint.2003.08.010. Amendt J, Campobasso CP, Gaudry E, Reiter C, LeBlanc HN, Hall MJR (2007) Best practice in forensic entomology—standards and guidelines. Int J Legal Med Conclusions 121:90–104. 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Egyptian Journal of Forensic SciencesSpringer Journals

Published: Jun 6, 2018

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