This is freely available on the journal website.The attached document is the pre-print/pre-refereeing [Author's original version] version of the article. This article has been accepted for publication in Journal of Medical Entomology, Vol.53(6), 2016, published by Oxford University Press. DOI: http://dx.doi.org/10.1093/jme/tjw0

Hall, MJR

Martin-Vega, D

Simonsen, TJ

Journal of Medical Entomology

Daniel Martín-Vega

Martin J. R. Hall

Thomas J. Simonsen

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Resolving Confusion in the Use of Concepts and Terminology in Intrapuparial Development Studies of Cyclorrhaphous Diptera

Developmental studies of necrophagous insects are strongly needed to support medico-legal investigations, because minimum postmortem intervals (min PMI) can be estimated from development data for species collected from a forensic scene together with accurate temperature information from that scene. The life cycle of cyclorrhaphous flies, which include some of the most used forensic indicators, shows an unusual feature as the pupal stage and the subsequent development of the pharate adult take place inside an opaque, barrel-like puparium, formed from the cuticle of the third-instar larva (Fraenkel and Bhaskaran 1973). Although unusual this feature is not unique among insects, or even among Diptera, despite the statement of Proença et al. (2014) . The period from pupariation (i.e., puparium formation) until the emergence of the adult is of special importance for forensic studies, as this period lasts for >50% of the total immature development. However, unlike the larval stage where a quantitative measure of age (e.g., body length) can be modeled in relation to time, the puparium shows virtually no external age-related changes (Amendt et al. 2011). Nevertheless, the puparium can be removed in order to determine morphological markers related to age on the insect inside, which can then be used for simple age estimation. Accordingly, a number of recently published studies (e.g., Pujol-Luz and Barros-Cordeiro 2012 , Defilippo et al. 2013 , Proença et al. 2014 , Ma et al. 2015) have described age-related morphological landmarks in the intrapuparial development of several forensically important Diptera, chiefly blow flies (Calliphoridae). Our concern has to do with confusion regarding concepts and terminology frequently occurring in these kinds of intrapuparial development studies. It is very likely that much of the existing confusion is related to the wide use of the terms “pupa,” referring to any fly individual during its intrapuparial development (regardless of which developmental stage lies inside the puparium), and “pupal stage” or “pupal period,” referring to the period from pupariation to adult emergence in forensic entomology (e.g., Amendt et al. 2011). This terminology might be practical but it is incorrect, as it includes within the “pupal period or stage” not only the actual pupal stage but also the prepupal stage and the final development of the pharate adult, even when the latter is significantly the longest intrapuparial stage in cyclorrhaphous flies  (Hinton 1971)

Martín Vega, Daniel

Hall, Martin J. R.

Simonsen, Thomas J.

e_Buah - Biblioteca Digital de la Universidad de Alcalá

1  Martín-Vega et al.: Terminology in 1 intra-puparial development studies 2  3 Journal of Medical Entomology 4 Letters to the Editor 5  6  7 Daniel Martín-Vega 8 Department of Life Sciences 9 Natural History Museum 10 Cromwell Road 11 SW7 5BD London 12 United Kingdom  13 Phone: +44 (0) 2079425770 14 e-mail: d.martin-vega@nhm.ac.uk 15  16 Resolving Confusion in the Use of Concepts and Terminology in Intra-Puparial 17 Development Studies of Cyclorrhaphous Diptera 18 Daniel Martín-Vega1, Martin J.R. Hall1 and Thomas J. Simonsen2 19  20 1Department of Life Sciences, Natural History Museum, SW7 5BD London, United 21 Kingdom 22 2Naturhistorisk Museum Aarhus, DK-8000 Aarhus C, Denmark 23 24 2  Developmental studies of necrophagous insects are strongly needed to support medico-25 legal investigations, because minimum post-mortem intervals (minPMI) can be estimated 26 from development data for species collected from a forensic scene together with 27 accurate temperature information from that scene. The life cycle of cyclorrhaphous 28 flies, which include some of the most used forensic indicators, shows an unusual feature 29 as the pupal stage and the subsequent development of the pharate adult take place inside 30 an opaque, barrel-like puparium, formed from the cuticle of the third-instar larva 31 (Fraenkel and Bhaskaran 1973). Although unusual this feature is not unique among 32 insects, or even among Diptera, despite the statement of Proença et al. (2014). The 33 period from pupariation (i.e. puparium formation) until the emergence of the adult is of 34 special importance for forensic studies as this period lasts for more than 50% of the total 35 immature development. However, unlike the larval stage where a quantitative measure 36 of age (e.g. body length) can be modelled in relation to time, the puparium shows 37 virtually no external age-related changes (Amendt et al. 2011). Nevertheless, the 38 puparium can be removed in order to determine morphological markers related to age 39 on the insect inside, which can then be used for simple age estimation. Accordingly, a 40 number of recently published studies (e.g. Pujol-Luz and Barros-Cordeiro 2012, 41 Defilippo et al. 2013, Proença et al. 2014, Ma et al. 2015) have described age-related 42 morphological landmarks in the intra-puparial development of several forensically 43 important Diptera, chiefly blow flies (Calliphoridae). Our concern has to do with 44 confusion regarding concepts and terminology frequently occurring in these kinds of 45 intra-puparial development studies. It is very likely that much of the existing confusion 46 is related to the wide use of the terms ‘pupa’, referring to any fly individual during its 47 intra-puparial development (regardless of which developmental stage lies inside the 48 puparium), and ‘pupal stage’ or ‘pupal period’, referring to the period from pupariation 49 3  to adult emergence in forensic entomology (e.g. Amendt et al. 2011). This terminology 50 might be practical but it is incorrect, as it includes within the ‘pupal period or stage’ not 51 only the actual pupal stage but also the prepupal stage and the final development of the 52 pharate adult, even when the latter is significantly the longest intra-puparial stage in 53 cyclorrhaphous flies (Hinton 1971).   54 Different authors have already highlighted the frequent confusion and misuse of 55 terminology in studies on the metamorphosis of cyclorrhaphous flies (e.g. Hinton 1946, 56 1971, 1973; Jenkin and Hinton 1966; Fraenkel and Bhaskaran 1973). Although the 57 readers can find more detailed descriptions in those publications, we believe that it is 58 worthwhile compiling a short review here of the correct terms for the most important 59 stages and events in the intra-puparial development to help reduce future confusion: 60 Pupariation refers to the formation of the puparium, it takes place when the contraction 61 of the post-feeding larva is irreversible and it is different from ‘pupation’ or formation 62 of the pupa, which takes place later. From pupariation to the first apolytic event the 63 insect should be called a prepupa, as it is still attached to the puparium (i.e. the larval 64 cuticle) (Fig. 1A–B). The term pupa should be used only when the larval-pupal apolysis 65 (i.e. the separation of the epidermal cells of the pupa from the larval cuticle or 66 puparium) is complete (Fig. 1C). At that time, the legs and wings have partially everted 67 but not the head; the morphology of the pupa still resembles that of the prepupa and it 68 should be called a cryptocephalic pupa (= “hidden head”) (Fig. 1C). Then, within 69 usually a relatively short period the head, legs and wings evert completely, and the 70 cryptocephalic pupa becomes the phanerocephalic pupa (“visible head”), where head, 71 thorax and abdomen are discernible (Fig. 1D). Shortly after the dramatic transformation 72 of the cryptocephalic into the phanerocephalic pupa, the pupal-adult apolysis (i.e. the 73 4  separation of the adult epidermal cells from the pupal cuticle) starts (Fig. 1E), and at its 74 completion the pupa has become the pharate adult (Fig. 1F), which will continue its 75 development, usually for several days, until its emergence from the puparium.  76 Given that the term ‘puparium’ is a paronym of ‘pupa’, it is not surprising that 77 there is frequent use of the latter as a malapropism. For example, Ma et al. (2015) give a 78 “morphological description of pupae” of the blow fly Chrysomya rufifacies (Macquart), 79 but what they actually describe is the morphology of the puparium. This malapropism 80 also affects other words derived from ‘pupa’ and ‘puparium’. For instance, Pujol-Luz 81 and Barros-Cordeiro (2012) suggest that obligatory parasitic flies show “a much larger 82 intra-pupal [sic] development”, but we assume they refer to either the whole intra-83 puparial period, as the title of their paper suggests, or to the actual pupal stage, with no 84 reference to the internal changes of the pupa. In the same way, Proença et al. (2014) 85 discuss “the developmental time of intrapupae [sic]” of different Chrysomya Robineau-86 Desvoidy species, although their study focuses on the external morphology of the 87 different intra-puparial stages. Also, in Proença et al. (2014) there is an account of the 88 pupariation process with a description of the gradual “acquisition of pigmentation of the 89 cuticle of the pupa” from white to black, which obviously refers to the darkening of the 90 puparium, i.e. the hardened cuticle of the third-instar larva, not the cuticle of the pupa. 91 Interestingly, Proença et al. (2014) write later that “after 66 h, the pupa showed gradual 92 body pigmentation” although, according to their results (and their statement just a few 93 lines above), at that time the insect is already the pharate adult, i.e. no longer a pupa. 94 This misuse of the term ‘pupa’ for every intra-puparial developmental stage generates 95 confusion and imprecise terms – like the ‘pupal morphogenesis’ of Ma et al. (2015), 96 which is actually mostly focussed on the adult morphogenesis – and classifications – 97 5  like the division into ‘juvenile’ and ‘mature pupa’ (Defilippo et al. 2013, Ma et al. 98 2014) even when both divisions would include part of the pharate adult stage. 99 Determining and classifying the intra-puparial developmental stages is another 100 frequent source of confusion and misinterpretation. Hinton (1971, 1973) convincingly 101 argued for the appropriateness of defining the intra-puparial stages of cyclorrhaphous 102 flies based on the apolyses rather than on ecdyses, as the larval-pupal apolysis is not 103 followed by a larval-pupal ecdysis. Indeed, the adult sheds both larval and pupal 104 cuticles simultaneously at emergence (Hinton 1973). A determination of the onset of 105 pupal and pharate adult stages that is not based on completion of the apolysis (e.g. 106 Defilippo et al. 2013, Proença et al. 2014, Ma et al. 2015) is therefore completely 107 arbitrary. Moreover, it must be highlighted that determining when an apolysis is 108 complete requires either histological (Fraenkel and Bhaskaran 1973) or virtual micro-109 computed tomographical sections (Fig. 1). Hence, the description of the larval-pupal 110 apolysis process by simple macroscopic examination of Chrysomya albiceps 111 (Wiedemann) prepupae by Pujol-Luz and Barros-Cordeiro (2012) is likely based on 112 misinterpretations of concepts. Determining the timing of the different intra-puparial 113 stages correctly and consistently is particularly crucial in forensic entomology, as a 114 misinterpretation of concepts may lead to errors in minPMI estimations. For example, 115 Defilippo et al. (2013) consider that the cryptocephalic pupal stage starts at the same 116 time as pupariation in Calliphora vicina Robineau-Desvoidy (compare Tables 1 and 2 117 in their paper). However, larval-pupal apolysis is not complete until several hours after 118 pupariation in this species (approximately 18 hours at 24 ºC; see Fig. 1C), therefore the 119 values determined by Defilippo et al. (2013) could lead to a significant error in minPMI 120 estimations.   121 6  It is not our aim to disregard the studies of Pujol-Luz and Barros-Cordeiro 122 (2012), Defilippo et al. (2013), Proença et al. (2014), and Ma et al. (2015). Indeed, we 123 are aware of the wide confusion regarding the terminology and concepts related to intra-124 puparial development and particularly in the forensic entomology literature (we have 125 probably also sometimes misused the terms) and therefore the reigning confusion is 126 understandable. We suggest replacing the widely and erroneously used ‘pupal stage’ 127 referring to the whole period from pupariation to adult emergence by ‘intra-puparial 128 period’ in forensic entomology literature. If ‘pupal stage’ is maintained because of its 129 wide use, it would be advisable to briefly mention that this term is used in a broader 130 sense, including the prepupal, pupal and pharate adult stages. Nevertheless, in the 131 particular case of intra-puparial development studies, the correct terminology should be 132 fastidiously applied as it is the only way of avoiding further confusion and 133 misinterpretations.     134  135 136 7  References cited 137 Amendt, J., C.S. Richards, C.P. Campobasso, R. Zehner, and M.J.R. Hall. 2011. 138 Forensic entomology: applications and limitations. Forensic Sci. Med. Pathol. 7: 379–139 392. 140 Defilippo, F., P. Bonilauri, and M. Dottori. 2013. Effect of temperature on six 141 different developmental landmarks within the pupal stage of the forensically important 142 blowfly Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae). J. Forensic 143 Sci. 58: 1554–1557. 144 Fraenkel, G., and G. Bhaskaran. 1973. Pupariation and pupation in cyclorrhaphous 145 flies (Diptera): terminology and interpretation. Ann. Entomol. Soc. Am. 66: 418–422. 146 Hinton, H.E. 1946. Concealed phases in the metamorphosis of insects. Nature 157: 147 552–553. 148 Hinton, H.E. 1971. Some neglected phases in metamorphosis. Proc. R. Ent. Soc. Lond. 149 C 35: 55–64. 150 Hinton, H.E. 1973. Neglected phases in metamorphosis: a reply to V.B. Wigglesworth. 151 J. Ent. (A) 48: 57–68. 152 Jenkin, P.M., and H.E. Hinton. 1966. Apolysis in arthropod moulting cycles. Nature 153 5051: 871. 154 Ma, T., J. Huang, and J.-F. Wang. 2015. Study on the pupal morphogenesis of 155 Chrysomya rufifacies (Diptera: Calliphoridae) for postmortem interval estimation. 156 Forensic Sci. Int. 253: 88–93. 157 8  Proença, B., A.C. Ribeiro, R.T. Luz, V.M. Aguiar, V.C. Maia, and M.S. Couri. 158 2014. Intrapuparial development of Chrysomya putoria (Diptera: Calliphoridae). J. 159 Med. Entomol. 51: 908–914. 160 Pujol-Luz, J.R., and K.B. Barros-Cordeiro. 2012. Intra-puparial development of the 161 females of Chrysomya albiceps (Wiedemann) (Diptera, Calliphoridae). Rev. Bras. 162 Entomol. 56: 269–272. 163 164 9  FIGURE CAPTIONS 165 Fig. 1. Micro-CT virtual sagittal sections of the blow fly Calliphora vicina Robineau-166 Desvoidy at different times after pupariation, stained in iodine 0.5M and scanned in a 167 Nikkon Metrology HMX ST 225 system (exposure: 500 ms; voltage: 110–130 kV; 168 current: 100 μA). (A) At pupariation, the prepupa is still attached to the puparium, i.e. to 169 the third-instar larval cuticle. (B) 12 hours after pupariation, larval-pupal apolysis is 170 taking place but it is still not complete, as the epidermis is still attached to the puparium 171 in some areas of the abdominal region (arrow). (C) 18 hours after pupariation, larval-172 pupal apolysis is complete as the epidermis has detached from the puparium over the 173 body (arrow); the legs and wings have partially everted and the prepupa has become the 174 cryptocephalic pupa. (D) 30 hours after pupariation, the head has everted (arrow) 175 although it will maintain a hyaline appearance until the migration of the fat bodies. The 176 cryptocephalic pupa has transformed into the phanerocephalic pupa. (E) 48 hours after 177 pupariation, the adult epidermis has detached from the pupal cuticle only in some areas; 178 the pupal-adult apolysis is still not complete. (F) 72 hours after pupariation, the pupal-179 adult apolysis is complete as the pupal cuticle has detached over the body; the insect is 180 now a pharate adult, i.e. no longer a pupa. Abbreviations: pc, pupal cuticle; ppm, 181 puparium. 182 10   183 

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This is freely available on the journal website.The attached document is the pre-print/pre-refereeing [Author's original version] version of the article. This article has been accepted for publication in Journal of Medical Entomology, Vol.53(6), 2016, published by Oxford University Press. DOI: http://dx.doi.org/10.1093/jme/tjw08NHM Repositor

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Resolving Confusion in the Use of Concepts and Terminology in Intrapuparial Development Studies of Cyclorrhaphous Diptera

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