Tropical Biomedicine 36(1): 131–142 (2019)

Boettcherisca peregrina (Diptera: Sarcophagidae): A flesh species of medical and forensic importance

Shang, Y.J.1, Lv, J.2, Wang, S.W.3, Ren, L.P.1, Chen, W.1 and Guo, Y.D.1* 1Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha 410013, Hunan, 2Department of Clinical Medicine, Xiangya School of Medicine, Central South University, Changsha 410013, Hunan, China 3Department of Forensic Science, School of Basic Medical Sciences, Xinjiang Medical University, Ürümqi 830011, China *Corresponding author e-mail: [email protected] Received 10 March 2018; received in revised form 28 November 2018; accepted 30 November 2018

Abstract. Boettcherisca peregrine, as a fly with the necrophagous habits found on human corpses and a vector of disease or parasitic, -producing agent, is a significant species in and medical context. This study reviewed the various aspects of this fly species, including morphology, bionomics, molecular analysis, medical and forensic entomology involvement, such as morphological characteristics of larva, puparia and adult, developmental rate of larvae, the effects of heavy metal (such as Cd and Cu) on the growth and developmentin of larvae, and the impact of some specific stimulis on the labellar chemosensory hair of B. peregrina. Species identification, gene and functions, myiasis and forensic case of this species were also outlined. Therefore, the paper has an important implication for improving the role of B. peregrina in medicine and forensic science.

INTRODUCTION (Majumder et al., 2012). Geographically, this fly has been found in many parts of Boettcherisca peregrina (Robineau- the world including Oriental, Palaearctic, Desvoidy, 1830) (Diptera: Sarcophagidae) is and Australasian regions (Wang et al., 2017). a significant flesh fly species for medical Boettcherisca.peregrina has been recorded and veterinary management, due to its ability in many countries of the Oriental region, such of causing myiasis in human and other as Thailand (Samerjai et al., 2014), India mammals as an ectoparasite, and potential (Sharma et al., 2015) and Malaysia (Tan et used to estimate the minimum postmortem al., 2010). Previous investigations on B. interval (PMImin) as colonizer in the early peregrina have focused on molecular stage of corpse decomposition in forensic analysis (Guo et al., 2010a), larval investigations (Byrd & Castner, 2009; morphology (Sukontason et al., 2010), using Greenberg, 1971; Wells et al., 2001). pteridine fluorescence to deduce the age of Additionally, the species is beneficial to the adults (Zhu et al., 2013), The influence of biosphere and many ecosystems as carrion drugs on flesh larval development (Goff, decomposers, due to their important role in 1991), the growth and development rate of the food chain (Ferrar, 1987). The adult of B. peregrina at various temperatures in B. peregrina are robust and large-sized different parts of the world (Wang et al., 2017), flies, with a red-tipped grid pattern on and cuticular hydrocarbon composition of abdomen, gray and black of the background. pupal exuviae for species (Ye B. peregrina has a pair of red eyes, and et al., 2007). Therefore, we review its the reproductive cycle is of larviparous morphology, bionomics, molecular analysis,

131 medical and forensic entomology involve- et al. (2010), Chen (2013) has provided ment to increase worldwide attention of this some features for species identification of sarcophagid species. B. peregrina larval. Additionally, Singh et al. (2012) summarised the characteristics Morphology that can be used for the larval identification One of the important tasks in forensic of common Sarcophagidae. Erzinçliog˘ lu entomology, medical, and veterinary (2007), Szpila (2010) has published several entomology is to identify the species significant papers on larval characters of involved, thus gathering the morphology Calliphoridae for species identification, information of all stages in B. peregrina’s life however, the characters used for cycle is the core for species identification distinguishing the larvae of sarcophagids (Sukontason et al., 2014). have not been critically revised. The life cycle of B. peregrina is Puparia are common remnants of carrion- comprises of larva, pupa and adult in breeding flesh flies which present on the accordance that the reproductive cycle of this decomposed cadavers involving forensic sarcophagids species is ovoviviparous investigations (Mazzanti et al., 2010). (Szpila et al., 2015) (Fig. 1). The larva of B. Samerjai et al. (2014) developed the key peregrina has three instars, each instar shed most commonly used to identify puparia of off its exuvium to transform into the next some flesh fly species, including B. instar (Majumder et al., 2012). Sukontason peregrina, B. nathani, L. pattoni, L.

Figure 1. The life cycle of B. peregrina.

132 ruficornis and S. dux. Meanwhile, Samerjai durations of B. peregrina was recorded et al. (2014) invented a cleaning apparatus in Suzhou (Wang et al., 2017), Beijing, for removing external sundries of the puparial Hangzhou, Guizhou (Chen, 2013) and with common laboratory appliance. It has Bangladesh (Majumder et al., 2012) (Table been summarised by some papers that 1). In addition, Goff (1993) found that the puparial features can be used for species various drugs have an effect on the identification (Sukontason et al., 2006). development of immature necrophagous For the adult of B. peregrina, it is only . As early as 1989 and 1991, Goff the adult males that can be identified with reported that the residues and metabolites certainty, with few suitable keys for of cocaine and heroin can accelerate the distinguishing the adult female of this development of the larvae of B. peregrina species (Smith, 1986; Wells et al., 2001). (Goff, 1989; Goff, 1991). Simultaneously, Goff The morphology key used to differentiate also emphasized the significance of further B. peregrina adult males from other analyses involving different classes of drugs, forensically important flesh flies has been concentrations, and necrophagous fly published (Chen, 2013; Lu, 2003). As for species (Goff, 1993). Thus, there is a clear other sarcophagid of medical importance, need for studying the impacts of different the characteristic to identify adult males of drugs on the developmental durations of P. ruficornis has been published in Thailand different carrion-breeding flies to establish (Chaiwong et al., 2009), while that for a systematic database for supporting identifying the South American genera was criminal investigations. updated (de Carvalho & de Mello-Patiu, Heavy metal pollution has severely 2008). Additionally, the review paper of the threatened people’s health and the biological S. dux and P. ruficornis also has been diversity and has become a global environ- reported (Suwannayod et al., 2013). mental problem (Sun et al., 2007). In the aspect of environmental risk assessment, the Bionomics flesh fly of B. peregrina is considered as a The developmental rate of carrion-breeding model organism to determine its response flesh fly is crucial to estimate the PMI in to heavy metal exposure, since they are forensic entomology (Wang et al., 2017). In commonly distributed in urban habitats and recent years, it has been emphasized to predominantly confronted with heavy metals gather precise development data in specific in polluted sites (Wu et al., 2013). The regions to improve the accuracy of PMImin metabolism and distribution of cadmium (Cd) (Amendt et al., 2011). The developmental in immature of B. peregrina have been

Table 1. Developmental times of life stages of B. peregrina at different constant temperatures

Developmental duration, h No. Temp, First- Second- Third- Pupal Total °C Wandering References instar instar instar stage duration

16 56.0±2.8 53.6±2.2 170.0±4.4 74.2±2.3 713.3±30.0 1064.7±34.8 19 40.5±5.3 43.0±2.0 121.3±4.7 61.3±7.2 490.0±16.2 756.0±19.0 22 29.0±1.0 28.6±3.0 95.2±1.8 40.0±2.8 366.8±2.7 559.6±5.5 Wang et 1 25 20.3±0.5 19.5±1.0 70.0±1.6 34.5±1.9 270.0±5.2 414.3±3.9 al., 2017 28 16.8±1.8 15.6±0.9 59.6±2.2 22.4±2.2 200.6±0.9 315.0±2.0 31 14.5±1.7 13.6±2.2 53.5±2.3 19.4±1.9 177.0±1.7 278.0±4.0 34 12.4±0.9 12.2±0.4 48.4±3.0 16.0±2.8 170.0±3.8 258.0±3.5

16 41 53 218 Unstated 648 960±40 20 19 40 133 Unstated 408 600±25 2 24 14 31 75 Unstated 258 378±15.75 Chen, 2013 28 13.5 25 57.5 Unstated 192 288±12 32 6.3 8 65.9 Unstated 172 252.2±10.5

133 reported (Aoki Y 1984). In addition, Wu et morphological ways have encountered many al. (2013, 2014) has reported the effects of challenges for identifying many females heavy metal for the metabolism, growth and immature stages of sarcosaprophagous and development, and the reproduction of species (Smith, 1986; Wells et al., 2001). B. peregrina. DNA-based method can supplement morpho- The chemosensory hairs of the B. logical identification by distinguishing peregrina is considered as a model system species credibly and rapidly with low in the aspect of studying invertebrate taste requirement for sample preservation (Guo reception, due to its simpler structure and et al., 2012a). mitogenome has been unique response to specific stimuli widely used for the research of species (Amakawa, 1990). Previous studies have identification (Harrison, 1989). The complete reported the effect of some specific stimulis mitochondrial genome of B. peregrina on the labellar taste receptor cells of B. (GenBank accession number: KF921296) peregrina, such as amiloride (Sadakata et al., has been sequenced by Zhong et al. (2016). 2002), multiple receptor sites (Furuyama Previous several studies had reported the et al., 1999), alkali metal ions (Kijima, 1997), species identification of the B. peregrina pH and several sugars (glucose, mannose, with other flesh flies. Such as the application sucrose, fructose, and maltose) in mixtures 465 bp fragments of mitochondrial cyto- and single solutions (Amakawa, 1990), G chrome c oxidase subunit I (COI) gene in India Protein Modulators (GTPyS and GDP~S) and (Sharma et al., 2015), the application 637 bp inositol 1,4,5-trisphosphate (IP3)-mediated cytochrome coxidase subunit II (COII) and transduction cascade (Koganezawa & 555 bp 16S rRNA fragments (Guo et al., Shimada, 2002a; Shimada, 1997). 2012b), the 189 bp fragments of COII gene This fly species lives closely related to (Guo et al., 2010a), the rDNA internal the human environment. B. peregrina was transcribed spacer 2 (ITS2) (Song et al., collected on rabbit carcasses in Malaysia 2008), the single nucleotide polymorphisms (Silahuddin et al., 2015), in Brazil (de Souza (SNPs) of the COII sequences (Zhang et al., & Von Zuben, 2016), in (Farrell et 2015a), the complete COI and COII gene al., 2015). In addition, Moribayashi, Ohtaki (Zhang et al., 2015b) in China. The species of and Kurahashi had done various researches B. peregrina and Sarcophag similis forms for the factors affecting development and two single clade respectively with high pupal diapause of B. peregrina (Moribayashi, support value of 72% in the NJ tree based on 2002), including the effects of photoperiodic a 272 base pair region of COI indicated the and arachidonic acid content on develop- similarity of these two species in China (Guo ment rates of the larvae of B. peregrina et al., 2010b). In addition, the identification (Moribayashi, 2016; Kurahashi & Ohtaki, power of the COI gene was evaluated using a 1979), the effects of geographic variation minibarcode region of 127 bp, standard (Moribayashi, 2001), different profiles of barcode region of 658 bp and the entire COI ecdysone secretion (Moribayashi, 1988), the region 1,535 bp on 99 species ring glands physiological differentiation of including B. peregrina (Jordaens et al., mature larvae (Moribayashi, 1992), chilling 2013). The differences within B. peregrina period (Moribayashi, 1999), the change of species were revealed based on a 278-bp ecdysone titer (Ohtaki, 1972), photoperiod segment of the COI gene and a 289-bp (Atsuko et al., 2008), pupal diapause and segment of the 16 ribosomal RNA (16S rDNA) nondiapause of B. peregrina. gene (Guo et al., 2011). The dangers of relying on a single gene Molecular analysis for species identification have been illustrated by recent researchs, while Species identification combing use of multiple genes is more Rapid and accurate species identification valuable for evolutionary analysis and is significant in biological sciences and species identification (Guo et al., 2014). To legal medicine (Cai, 2010). Traditional raise the identification efficiency of certain

134 genes, the molecular markers are required oleae), the common house fly (M. domestica) to be further screened and optimized. and the Mediterranean fruit fly (Ceratitis Meanwhile, it is necessary to explore capitata) (Dubendorfer et al., 2002). Agrawal accurate, rapid and reliable species et al. (2010) reported that B. peregrina have identification methods that have relatively 12 chromosomes, including 5 pairs of meta/ low requirement to the samples preservation submetacentric autosomes and a pair of small so as to improve the application of flesh dot-like sex chromosomes-XY in the males flies in forensic investigation. and XX in the females. Some genes are involved in the sex determination pathway Gene and functions in dipteran insects, including the daughterless The gene of provides rich information (da), doublesex (dsx), sex lethal (sxl), for studying the mechanisms of anti- transformer (tra), transformer 2 (tra2), microbial peptides, sex-determination, maleless (mle), and fruitless (fru) (Andere chemoreceptors, and insecticide resistance et al., 2016). (Adams et al., 2000). The metabolism and xenobiotic resis- Many chemoreceptors and associated tance, detoxification genes have been proteins played a very important role in the reported in P. regina (Andere et al., 2016), gustatory and olfactory abilities of insects, in M. domestica (Scott et al., 2014), in D. these proteins were encoded by at least four melanogaster (Adams et al., 2000). For B. major gene families (Touhara & Vosshall, peregrina, these aspects remain undefined 2009). Koganezawa and Shimada (2002b) and needs to be studied in the future. The reported a cDNA library of taste tissue of the genome sequence of B. peregrina was B. peregrine. They found seven OBP genes expected to provide more information about named gustatory PBP-related proteins this species in the relationship between gene (GPBPRPs) 1-7, the GPBPRP6 and 7 genes and proteins functions. were expressed mainly in labellum, and GPBPRP2, 3 and 5 genes were expressed in Myiasis tarsus, which reflecting different functions in Myiasis is caused by the larvae of necro- different taste tissues. phagous flies invaded the organs and tissues In general, insects have a diverse innate of warm-blooded vertebrate and immunity pathway to inhibit the survival of humans (Ren et al., 2018). Those parasites various microbes (Stokes et al., 2015), and to larvae mainly stayed in the mouth, eyes, resist bacterial and fungal , which nose, subcutaneous tissues, skin, stomach, can activate an antimicrobial defense system intestines, urinogenital system, ears, and by expressing the related genes (Ferrandon other soft tissues of the host (Hall et al., 2016). et al., 2007). Ando (1987) purified three The human myiasis caused by B. peregrina antibacterial proteins from the hemolymph was very rare and, only several myiasis of B. peregrina third instar larvae named cases available, which occurred in different sarcotoxin IIA, IIB, and IIC. Natori (2010) sites, such as nasal (Kamimura, 1986), oral discussed and summarized the functions of (Matsuzaki, 1987) and the eyes (Miura et al., the immune molecules in the hemolymph of 2005). Such cases suggest that proper control B. peregrina larvae, and especially measures for flies involved in myiasis in emphasised the dual roles of some immune humans are necessary. In addition to the molecules in development and defense, medical importance of flesh flies as including antibacterial proteins, humoral mechanical vectors of parasitic disease lectin, antifungal protein (AFP) and small agents, they can cause myiasis in the hospital antibacterial compound (5-S-GAD). environment which are also called noso- Typically, the heteromorphic XX/XY comial myiasis, and can be considered as an system determined the sex of insects, and indicator of wound care neglect, either by the Y-linked is male determining genes (Andere nurses or by oneself (Nazni, 2011). Miura et al., 2016). This phenomenon has been et al. (2005) reported that the larvae of B. confirmed in the olive fruit fly (Bactrocera peregrina were found at the left conjunctival

135 sac of a patient in a Japan hospital, which difficulties in identifying the larvae or adults further confirmed the potential of this species of sarcophagid species associated with in nosocomial invasion. human remains (Sukontason et al., 2014; Suwannayod et al., 2013). In addition, the Forensic entomology practical application of necrophagous The role of forensic entomology in forensic insects in forensic investigations indicated investigation has become increasingly that the developmental duration and local important. The larvae and adults of necro- succession data is necessary, as the more phagous insects found on decomposed similar between the experiment and the real corpses can provide significant information case in climatic and micro-environmental for PMI estimations, especially for the conditions, the more accurate the PMI can corpses with intervals more than 72h (Byrd be obtained (Wang et al., 2017). On the other & Castner, 2009; Cai, 2010). Case studies have hand, great efforts are needed by the forensic been described in research articles (Arnaldos scholars to introduce standards protocols et al., 2005), books (Cai, 2010), which when handling cases in order to determine demonstrated the ability of forensic the time of death accurately (Ying et al., entomology to accurately estimate the PMI. 2013). Boettcherisca peregrina is one of the dominant species in some case studies and the most widespread sarcophagid species CONCLUSIONS found on human corpses in China (Chen, 2013; Wang et al., 2017). The potentiality of B. Although the information of B. peregrina peregrina for estimating the PMI in forensic is relatively limited, the importance of entomology has been well demonstrated by this sarcophagid species is improving, many studies, such as in Switzerland (Cherix particularly in the area of forensic et al., 2012), in Thailand (Sukontason et al., entomology. In addition to the PMI estimation, 2007), in Hawaiian Islands (Goff, 1987). In further exploring of necrophagous habits China, Ying et al. (2013) reported that a of B. peregrina in view of the role in decomposing female corpse in Xiang River carrion decomposition such as the potential of Hunan province was found, and the main mechanisms driving the colonization patterns larvae found on the corpse were identified is also important. This study reviewed as B. peregrina by both morphologic various aspects of this fly species, including observation and mitochondrial DNA morphology, bionomics, molecular analysis, sequence. PMI was estimated between 90 medical and forensic entomology involve- and 120 hours based on the entomological ment. However, further study on various evidence of experimentally obtained. bionomic of this species is necessary, such However, according to the forensic investi- as developmental rates in fluctuating gation, the interval was up to 128 hours. temperature conditions, thermal parameters, Although the knowledge of local fauna, lower developmental threshold, the relevant climatic and micro-environment had been factors effecting development durations, considered in this case, the PMI was flight activity, seasonal prevalence and all inaccurately estimated. This case indicated aspects involved in the application in that ecological, evolutionary, and genetic medical and forensic entomology. The mechanisms of carrion decomposition molecular markers for species identification require further investigation (Amendt et al., are still required to be further screened 2011; Smith, 1986). and optimized. Gene and functions of B. Compared with blowflies, flesh flies are peregrina need to be studied further in the rarely applied in forensic investigations to future to understand the mechanisms of the estimate the PMI, despite they have obvious evolution, population structure, behavior, and advantages for decomposed corpses (Cherix physiology of this species. Although such et al., 2012), which is mainly due to the studies are time-consuming, efforts including

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