J Forensic Sci, May 2015, Vol. 60, No. 3 doi: 10.1111/1556-4029.12707 Available online at: onlinelibrary.wiley.com
CASE REPORT PATHOLOGY/BIOLOGY Alejandro Gonzalez Medina,1 M.Sc.; Oscar Soriano Hernando,2 Ph.D.; and Gilberto Jimenez Rıos,1 M.D., Ph.D.
The Use of the Developmental Rate of the Aquatic Midge Chironomus riparius (Diptera, Chironomidae) in the Assessment of the Postsubmersion Interval
ABSTRACT: Nonbiting midges (Diptera, Chironomidae) are the most abundant members of the fauna associated with submerged carcasses, but their use in the medicolegal context is very restricted because of their complex ontogeny. In this case, the corpse of a woman was recovered in late spring from a river in Granada (Iberian Peninsula). It showed obvious signs of long permanence in the aquatic environment and, along with pulmonary and microscopical analyses, led to the conclusion that the cause of death was drowning. Several larvae-like specimens were sampled from the scalp and later identified by morphological external features as IV instar larvae of Chironomus riparius Meigen, 1804 (Diptera, Chironomidae). Sequencing of cytochrome oxidase subunit I was performed to confirm the identification. The knowledge of the biology of C. riparius at low temperatures was critical to assess a postsubmersion interval of 16–17 days.
KEYWORDS: forensic science, forensic entomology, Chironomidae, Chironomus riparius, postmortem interval, postmortem changes, drowning
The access of human beings to watercourses and their use can lead to the genesis of many situations where the outcome can be harmful or fatal, with drowning as one of its main consequences. Drowning was classically defined as that form of respiratory impairment which results from submersion/immersion in a liquid medium, usually water (1,2). Although in general terms this definition is accepted, drowning is not merely a pathophysiological entity and some cases fall outside these conceptual boundaries (3). It is one of the most problematic forms of violent death from a medicolegal point of view because of the large variability of its manifestations in terms of endogenous factors and environmental features. Such complexity is manifested mainly at three levels: cause of death, medicolegal etiology and time of death. Forensic entomology makes available to forensic investigators a set of techniques based on the study of developmental biology and dynamics of invertebrate communities to resolve legal issues (4). The most frequent medicolegal issue that requires the assistance of a forensic entomologist is the time of death assessment, given that entomological estimates are more accurate than biochemical or physiological methods from 72 h after death (5). A corpse exposed in terrestrial environment in optimal conditions attracts many sarcosaprophagous fauna members. Their develop1 Institute of Legal Medicine of Granada, Avenida de la Innovacion, 1, 18007, Granada, Spain. 2 Department of Biodiversity and Evolutive Biology, Museo Nacional de Ciencias Naturales (CSIC), Calle Jose Gutierrez Abascal, 2, 28006, Madrid, Spain. Received 26 Sept. 2013; and in revised form 12 April 2014; accepted 27 April 2014.
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mental rates and colonization sequence on decaying organic matter provide much information about the interval comprised between the arrival time of primary colonizers (the first arthropods colonizing the body) and the time of analysis, known as period of insect activity (PIA) (6). The earlier the colonization after death by the primary colonizers, the closer the PIA estimates postmortem interval (PMI). It should be noted that not all cases fit the aforementioned standard and occurrence of physical barriers that impede access of insects to the corpse is common (7). One of the most common physical barriers is the immersion of the body in a liquid medium, which prevents colonization of the corpse by the typical entomological fauna of exposed bodies in terrestrial settlements. However, this preventive action does not eliminate the possibility of the corpse utilization as a resource by the aquatic fauna. The role of benthic arthropods in the cadaveric ecosystem is not well known and is still a wide open field of research (8). Chironomidae flies (Diptera) are one of the most ubiquitous and usually the most abundant insects in freshwater environments worldwide. Larvae and pupal instars live in all types of freshwater settlements (all freshwaters bodies, including Arctic region, highest mountains, deepest lakes as Lake Baikal, phytotelmata, and thermal springs), some chironomids tolerate high osmotic levels and some genera include littoral and marine species (9). Currently, 10,000 species are described (10). Identification of chironomids is mainly based on larvae, pupae and imago morphological features as it is of many other groups of insects. However, larval instars of chironomids, commonly collected in aquatic samplings, surveys, etc., show few morphological features useful for identification (11), and it frequently depends on © 2015 American Academy of Forensic Sciences
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association of those instars with identified pupal exuviae or adult males which tend to have more species-specific characteristics. In this case, the knowledge about the development of Chironomus riparius Meigen 1804 (Diptera, Chironomidae) was applied to establish the postmortem submersion interval (PMSI) in a case of drowning in a zone of the Genil River belonging to the urban waters of Granada Province (Spain, SE Iberian peninsula). The singularities and use of this component of the sarcosaprophagous aquatic fauna are subsequently discussed. Case Description and Methodology In June 2, 2013, the corpse of a woman was found floating on the Genil River on its way through the city of Granada (UTM WGS84 coordinates, X: 447102.43, Y: 4113715.98; zone 30, Northern Hemisphere). Members of the Policıa Nacional attended to the site of the discovery and reported that the identity of the victim could correspond to that of a woman with a psychiatric record, who had disappeared 17 days earlier. According to the statements from several witnesses present at the time of the disappearance, it was most likely a suicide, but they were not able to establish the exact point of entry into water. The corpse was taken to the Institute of Legal Medicine of Granada, where an autopsy was performed in accordance with current European regulations (12). The entomological evidence was collected by the coroner in charge of the autopsy according to the standards and guidelines published by the European Association for Forensic Entomology (EAFE) applicable to the terrestrial cadaveric fauna (6) as specific guidelines for aquatic fauna were not considered in that protocol. It was also registered the water temperature at the time of the corpse removal from the water (13°C). One day after the autopsy, some points of the river bottom around the discovery zone were sampled with a dipper. Regarding morphological identification, external features were observed with the aid of a dissecting microscope. After proteinase K tissue digestion, head capsules slides were mounted in DMHF (dimethylhydantoin formaldehyde resin). The specimen identifications were performed using specific taxonomic keys (13,14). To confirm the morphological identification and discriminate among related or cryptic species, total DNA was extracted from body of larvae using the Bio Sprint 15 DNA kit (Qiagenâ GmbH, Germany). Partial sequences of cytochrome oxidase subunit I (COI) were amplified by polymerase chain reaction (PCR) using the primers LCO1490 and HCO2198 (15). The PCR mix contained 2 lL DNA, 2.5 lL of the corresponding buffer, 1.5 lL MgCL2 (25 mM), 0.5 lL dNTPs mix (10 mM each), 1.25 lL of both primers (10 lM), 0.3 lL Taq DNA polymerase (5 U/lL) (Promegaâ Madison, WI, USA), and ddH2O for a total volume of 25 lL. The following PCR conditions were used in the amplifications: 94°C (4 min); 5 cycles of 94°C (45 sec), 40° (1 min), 72° (1 min); 35 cycles of 94°C (45 sec), 44° (1 min), 72° (1 min); and a final extension at 72° (10 min). The products were visualized with blue light on 0.8% agarose gels stained with SYBR Safe (Invitrogenâ Carlsbad, CA, USA) with comigrating 100 bp:1 kb ladder molecular weight markers to confirm the correct amplification. The amplified fragments were purified by ethanol precipitation prior to sequencing both strands using BigDye Terminator kits (Applied Biosystems, ABIâ Foster City, CA, USA). Products were electrophoresed on ABI 3730 genetic analyser (Applied Biosystemsâ). The forward and reverse DNA sequences obtained for each specimen were
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aligned and checked using the Sequencher software (Gene Codes Corporationâ Ann Arbor, MI, USA.) after removing primer regions. To verify the species identification, a BLAST analysis was conducted.
Results Autopsy Findings The body was in initial putrefactive conditions, with widespread green staining especially intense on the chest and neck (Fig. 1). Apart from the putrefactive dermo-epidermal detachment, a very intense maceration was noted, mainly in hands and feet. The corpse was covered by a single layer of clothing, irregularly smeared with mud compatible with the bottom of the river. No signs of external violence were observed. Given that the corpse was discovered floating and no nail detachment or evisceration was observed, the decay stage was classified as “early floating” according to Haefner’s description of submerged carcasses (16). The macroscopic internal examination was severely limited because all organs and systems were affected in varying degrees by decomposition. The weights of the most relevant organs are summarized in Table 1. The macroscopic findings on the respiratory system, stomach, and duodenum were remarkable: incipient Paltauf’s spots were observed in lungs accompanied by internal hemorrhagic suffusions evidenced when cut and intense congestion of trachea and bronchi. The respiratory airways were clean and no tracheobronchial foam fungus was observed. In the stomach and duodenum, atrophic mucosa and a water content exceeding 500 mL were easily observable.
FIG. 1––Head and neck of the corpse. Note the generalized green stain, detachment of the skin and mud smears.
TABLE 1––Weight of most relevant organs examined during the autopsy. Body area Thorax Abdomen
Organ
Weight (g)
Right lung Left lung Heart Spleen Right kidney Left kidney
405 345 275 100 125 120
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The comparative analysis of diatoms observed in closed viscera and the water recovered from the place of the corpse removal, histological findings and serological values of strontium (17,18) were consistent with a death by drowning. Entomological Findings The only entomological evidence collected during the autopsy was interposed among the long hairs of the head, in the area close to the scalp. They presented vermiform appearance, but with a distinct head capsule (eucephalic larvae) (Fig. 2A), ventrally arranged mouthparts (Fig. 2B) and thoracic appendages or prolegs along with lateral tubules in terminal abdomen (Fig. 2C), characteristic features of the Chironomidae family. A row of radially arranged grooves from mandible (Fig. 2D), typical from Chironomus larvae, was observed. Morphological analysis led to conclude that the specimens recovered from the autopsy were IV instar larvae of C. riparius. Water temperature in the moment of the corpse recovery was 13°C and during the following days later it did not change. No larvae with external morphology compatible with the previously mentioned species were captured during the river sampling near the corpse location. One of three sequences obtained (658 bases pairs) matched exactly the C. riparius sequences found in GenBank (e.g. accession numbers HM137887—coverage 100%, HM137893 and HM137888—coverage 99%) (19). The other two, which also blasted to C. riparius (in their case, with an identity of 99%), showed only one substitution with respect to the first one in the position 271. These results confirmed the presumptive morphological identification. Discussion The time of death is a major medical–legal problem faced by forensic specialists in daily practice. Immersion of a body in a liquid medium adds new difficulties to those problems already present when attempting to estimate the postmortem interval
from macroscopic or biochemical evidence. Factors such as low temperatures, currents, or pollution can introduce variations that hinder the interpretation of analytical results. Foam fungus is an important marker of vital drowning, although it is not present in all cases where this mechanism of death occurs (20). In this one, the diagnosis of that sign was negative. One possible explanation for this absence is the considerable lapse of time between drowning and the corpse discovery, which may have facilitated the washing away of the airways and mucous clearance. The study of PMSI has been classically addressed by comparing the information from the current case with casuistic repositories with known PMSI. Reh’s tables from data registered in Germany are some of the most used in our region (21,22), but their use should be undertaken with caution. The main limitations of this system based on the analogy are the differences in month/temperature with the case under study and biogeographic particularities. Correction factors should be applied depending on causal circumstances found in the biogeographic zone where the model will be implemented and endogenous factors should be included (such as weight and age) to minimize variations and errors in the model (23). Regression formulae derived from quantification of potassium from vitreous humor have been used for drowning cases (24). The main problems with this technique are the integrity of the eyeball, rarely kept because of the action of fishes and macroinvertebrates, and the erosive action of obstacles during passive drag. Another biochemical method, consisting in quantification of chlorophyll a levels, has proved very effective results in PMSI estimation in both pig carcasses and inanimate objects such as ceramic tiles (16). Despite the undeniable interest of these techniques, developmental parameters of C. riparius were actually used to calculate PMSI because they have never been tested before on a forensic case and their use could be extrapolated to similar settings. The advantages offered by the use of Chironomidae as forensic indicators of time of death are considerable (25). On one hand, it is an external indicator, so that it is not subject to the
FIG. 2––Chironomus riparius morphological details (A) whole larva; (B) mentum, ventral view; (C) ventral tubules on 11th abdominal segment, lateral view; (D) mandible, lateral view, BR: basal row.
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vicissitudes of the body that may occur as a result of postmortem scavenging or other alterations. Furthermore, the early arrival of these insects to the submerged corpse (26,27) would favor the use of the same techniques used with primary terrestrial colonizers, based on the study of development of pre-imaginal stages. Phenological studies of this species conducted in other biogeographic areas have concluded that IV instar larvae were sampled in the water from March to November, completing 4–7 generations (28). Such data have no interest to our case, as the body was found in early June, which gives us an interval with a margin of error from March to June. It is therefore necessary to use either successional data or, if Chironomidae are considered as early colonizers, ontogenic development data. Most of the Chironomidae life cycle takes place in an underwater environment. The complete metamorphosis requires from a minimum of 1 week up to several years depending on the species, water temperature, and nutrient availability among other factors (29). In the particular case of C. riparius, larvae stay inside of an U-shaped tunnel about 10 mL deep, dug into the river bottom. From the tunnel, they feed on suspended particulate organic matter that is deposited on the tunnels (30). The discovery of C. riparius in the hair of the deceased may be explained given the degradative behavior of hair keratins, biochemically similar to the river bottom. For river systems, the direct contact with the medium implies that the same type of biome has many factors that affect more directly the development and faunal diversity that in the case of terrestrial ecosystems: degree of eutrophication, oxygen, food availability, temperature water, pH, and flow speed. Because of the high environment specificity of certain species (31), these can be used not only as an indicator of PMSI, but also displacement of the body. Chironomus riparius is almost cosmopolitan, able to live in waters with a moderate to high level of contamination (32), pH between 4–8 (33), and low oxygen levels (34). All these features are compatible with the site of the discovery of the corpse. Perhaps one of the biggest difficulties that must be considered is the behavior of Chironomidae in diapause (arrested development due to unfavorable environmental conditions). Terrestrial Diptera are sensitive mainly to both temperature decrease and, below a certain value (called basal temperature or lower developmental threshold), the length increase as metamorphosis progress is arrested at any stage of development. In Chironomidae, not all instars enter into diapause; for example, it is known that C. riparius usually enters in diapause in the larval instar IV, but instar III can also enter when photoperiod and temperature conditions are suitable (35). Also, diapause disturb imaginal disks development (tissue systems which give rise to adult structures), but it does not stop the growth of the individual length, allowing to observe higher individuals than the largest individuals that developed in optimum temperatures. The maximum length observed in the sampled specimens was 15 mm, which is obviously a larger result compared with the maximum that the same species reaches when breeding under controlled temperature above the basal temperature (12.6 mm at 14°C) (36), which suggests that development was below this threshold. The absence of retrospective records of water temperature impeded more accurate estimates. If, as some studies suggest, Chironomidae larvae colonize the corpse as soon as terrestrial primary colonizers do (8,27), it can be known how long the corpse remained, at least, in the water.
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Always bear in mind that there is a period of uncertainty (and therefore not measurable) between the submersion and the arrival of the various associated species. There are many works on the development of immature stages of C. riparius as it is a classic indicator of pollution from different types of water and has been extensively studied. From previously published studies, it is known that: • If we apply Mackey’s formula (37), duration from larva I to pupa at 13°C is 27.1 days. • Goddeeris and collaborators found out in an experiment carried out at Belgium that 50% of larvae develop to IV instar at 13°C in 14 days (35). In this case, all larvae were IV instar, so 14 days would be a too conservative estimation. • In laboratory controlled conditions (36), it was recorded that, at 15°C, larvae develop to pupae in 16 days. It must be taken in consideration that environmental temperatures in this case were below 15°C, so development duration of larvae may be larger. Considering those references that set the lower (36) and upper limit (37) for Chironomidae development under specific temperature regimes, C. riparius activity on the corpse had to begin 16–27.1 days before the corpse recovery (in the refrigeration chamber, given the extremely low temperatures and the absence of water, Chironomidae do not grow), that is, between May 5–6, 2013 and May 18, 2013. If we accept the interval of disappearance as true (May 17, 2013 to June 2, 2013), submersion took place most probably in the time range when PIA and the disappearance match, that is, between May 17, 2013 and May 18, 2013. All the particularities explained above vary depending on the species, so accurate identification of specimens collected is essential to draw conclusions from real casework. DNA bar coding relies on sequence variation in short fragments of DNA to serve as a unique species identifier. Variations in the sequence of cytochrome c oxidase subunit I (COI) have proven useful for the identification of many animal taxa, including insects (38), members of the family Chironomidae (39,40), and Chironomus species (11,41). Difficulties in the taxonomy of Chironomus species are due to the existence of large complexes of cryptic species, which can be best distinguished by the banding pattern of polytene chromosomes (42). Cytological characterization, however, requires considerable expertise and is also time consuming, so molecular-based approaches, such as DNA bar coding, are being used to supplement traditional taxonomic methods of species identification. This case is a clear example that a detailed knowledge of the developmental biology of the Chironomidae can shed light on questions that otherwise might be answered only circumstantially or with higher error margins. The number of aquatic species about which we know details applicable to forensic practice are few and almost always phenological data offers too broad margins. Protocols for collecting and interpreting evidence for aquatic fauna are necessary to harmonize practices and transfer results in the context of forensic practitioners. Acknowledgments We would like to thank Annie Machordom, senior researcher and laboratory chief at the Museo Nacional de Ciencias Naturales, for providing the technical resources for the realization of molecular biology analysis.
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24. Bray M. Chemical estimation of fresh water immersion intervals. Am J Forensic Med Pathol 1985;6:133–9. 25. Haskell NH, McShaffrey DG, Hawley DA, Williams RE, Pless JE. Use of aquatic insects in determining submersion interval. J Forensic Sci 1989;34:622–32. 26. Merritt RW, Wallace JR. The role of aquatic insects in forensic investigations. In: Byrd JH, Castner JL, editors. Forensic entomology: the utility of arthropods in legal investigations. Boca Raton, FL: CRC Press, 2010;271–320. 27. Vance GM, Vandyk JK, Rowley WA. A device for sampling aquatic insects associated with carrion in water. J Forensic Sci 1995;40:479–82. 28. Groenendijk D, Postma JF, Kraak MHS, Admiraal W. Seasonal dynamics and larval drift of Chironomus riparius (Diptera) in a metal contaminated lowland river. Aquat Ecol 1998;32:341–51. 29. Coffman WP, Ferrington LC. Chironomidae. In: Merritt RW, Cummins KW, editors. An introduction to the aquatic insects of North America. Dubuque, IA: Kendall Hunt, 1996; 551–652. 30. Stief P, de Beer D. Probing the microenvironment of freshwater sediment macrofauna: implications of deposit-feeding and bioirrigation for nitrogen cycling. Limnol Oceanogr 2006;51(6):2538–48. 31. Moller Pillot HKM. Chironomidae larvae II - biology and ecology of the Chironomini, 1st edn. Zeist, Netherlands: KNNV, 2009. 32. Davies LJ, Hawkes HA. Some effects of organic pollution on the distribution and seasonal incidence of Chironomidae in riffles in the River Cole. Freshwater Biol 1981;11:549–59. 33. Palawski DU, Hunn JB, Chester DN, Wiedmeyer RH. Interactive effects of acidity and aluminium exposure on the life cycle of the midge Chironomus riparius (Diptera). J Freshwater Ecol 1989;5:155–62. 34. Penttinen OP, Holopainen IJ. Physiological energetics of a midge, Chironomus riparius Meigen (Insecta, Diptera): normoxic heat output over the whole life cycle and response of larva to hypoxia and anoxia. Oecologia 1995;103:419–24. 35. Goddeeris BR, Vermeulen AC, De Geest E, Jacobs H, Baert B, Ollevier F. Diapause induction in the third and fourth instar of Chironomus riparius (Diptera) from Belgian lowland brooks. Arch Hydrobiol 2001;150:307–27. 36. Pery ARR, Garric J. Modelling effects of temperature and feeding level on the life cycle of the midge Chironomus riparius: an energy-based modelling approach. Hydrobiol 2006;553:59–66. 37. Mackey AP. Growth and development of larval Chironomidae. Oikos 1977;28:270–5. 38. Meier R, Shiyang K, Vaidya G, Ng PK. DNA barcoding and taxonomy in Diptera: a tale of high intraspecific variability and low identification success. Syst Biol 2006;55(5):715–28. 39. Pfenninger M, Nowak C, Kley C, Steinke D, Streit B. Utility of DNA taxonomy and barcoding for the inference of larval community structure in morphologically cryptic Chironomus (Diptera) species. Mol Ecol 2007;16(9):1957–68. 40. Guryev V, Makarevitch I, Blinov A, Martin J. Phylogeny of the genus Chironomus (Diptera) inferred from DNA sequences of mitochondrial cytochrome b and cytochrome oxidase. Mol Phylogenet Evol 2001;19 (1):9–21. 41. Carew ME, Pettigrove V, Hoffmann AA. Identifying chironomids (Diptera: Chironomidae) for biological monitoring with PCR-RFLP. Bull Entomol Res 2003;93(6):483–90. 42. Martin J. Chromosomes as tools in taxonomy and phylogeny of Chironomidae (Diptera). Entomol Scand 1979;10:67–74. Additional information and reprint requests: Alejandro Gonzalez Medina, M.Sc. Institute of Legal Medicine of Granada Avenida de la Innovacion, 1 18100 Granada Spain E-mail: [email protected]
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CASE REPORT PATHOLOGY/BIOLOGY Alejandro Gonzalez Medina,1 M.Sc.; Oscar Soriano Hernando,2 Ph.D.; and Gilberto Jimenez Rıos,1 M.D., Ph.D.
The Use of the Developmental Rate of the Aquatic Midge Chironomus riparius (Diptera, Chironomidae) in the Assessment of the Postsubmersion Interval
ABSTRACT: Nonbiting midges (Diptera, Chironomidae) are the most abundant members of the fauna associated with submerged carcasses, but their use in the medicolegal context is very restricted because of their complex ontogeny. In this case, the corpse of a woman was recovered in late spring from a river in Granada (Iberian Peninsula). It showed obvious signs of long permanence in the aquatic environment and, along with pulmonary and microscopical analyses, led to the conclusion that the cause of death was drowning. Several larvae-like specimens were sampled from the scalp and later identified by morphological external features as IV instar larvae of Chironomus riparius Meigen, 1804 (Diptera, Chironomidae). Sequencing of cytochrome oxidase subunit I was performed to confirm the identification. The knowledge of the biology of C. riparius at low temperatures was critical to assess a postsubmersion interval of 16–17 days.
KEYWORDS: forensic science, forensic entomology, Chironomidae, Chironomus riparius, postmortem interval, postmortem changes, drowning
The access of human beings to watercourses and their use can lead to the genesis of many situations where the outcome can be harmful or fatal, with drowning as one of its main consequences. Drowning was classically defined as that form of respiratory impairment which results from submersion/immersion in a liquid medium, usually water (1,2). Although in general terms this definition is accepted, drowning is not merely a pathophysiological entity and some cases fall outside these conceptual boundaries (3). It is one of the most problematic forms of violent death from a medicolegal point of view because of the large variability of its manifestations in terms of endogenous factors and environmental features. Such complexity is manifested mainly at three levels: cause of death, medicolegal etiology and time of death. Forensic entomology makes available to forensic investigators a set of techniques based on the study of developmental biology and dynamics of invertebrate communities to resolve legal issues (4). The most frequent medicolegal issue that requires the assistance of a forensic entomologist is the time of death assessment, given that entomological estimates are more accurate than biochemical or physiological methods from 72 h after death (5). A corpse exposed in terrestrial environment in optimal conditions attracts many sarcosaprophagous fauna members. Their develop1 Institute of Legal Medicine of Granada, Avenida de la Innovacion, 1, 18007, Granada, Spain. 2 Department of Biodiversity and Evolutive Biology, Museo Nacional de Ciencias Naturales (CSIC), Calle Jose Gutierrez Abascal, 2, 28006, Madrid, Spain. Received 26 Sept. 2013; and in revised form 12 April 2014; accepted 27 April 2014.
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mental rates and colonization sequence on decaying organic matter provide much information about the interval comprised between the arrival time of primary colonizers (the first arthropods colonizing the body) and the time of analysis, known as period of insect activity (PIA) (6). The earlier the colonization after death by the primary colonizers, the closer the PIA estimates postmortem interval (PMI). It should be noted that not all cases fit the aforementioned standard and occurrence of physical barriers that impede access of insects to the corpse is common (7). One of the most common physical barriers is the immersion of the body in a liquid medium, which prevents colonization of the corpse by the typical entomological fauna of exposed bodies in terrestrial settlements. However, this preventive action does not eliminate the possibility of the corpse utilization as a resource by the aquatic fauna. The role of benthic arthropods in the cadaveric ecosystem is not well known and is still a wide open field of research (8). Chironomidae flies (Diptera) are one of the most ubiquitous and usually the most abundant insects in freshwater environments worldwide. Larvae and pupal instars live in all types of freshwater settlements (all freshwaters bodies, including Arctic region, highest mountains, deepest lakes as Lake Baikal, phytotelmata, and thermal springs), some chironomids tolerate high osmotic levels and some genera include littoral and marine species (9). Currently, 10,000 species are described (10). Identification of chironomids is mainly based on larvae, pupae and imago morphological features as it is of many other groups of insects. However, larval instars of chironomids, commonly collected in aquatic samplings, surveys, etc., show few morphological features useful for identification (11), and it frequently depends on © 2015 American Academy of Forensic Sciences
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association of those instars with identified pupal exuviae or adult males which tend to have more species-specific characteristics. In this case, the knowledge about the development of Chironomus riparius Meigen 1804 (Diptera, Chironomidae) was applied to establish the postmortem submersion interval (PMSI) in a case of drowning in a zone of the Genil River belonging to the urban waters of Granada Province (Spain, SE Iberian peninsula). The singularities and use of this component of the sarcosaprophagous aquatic fauna are subsequently discussed. Case Description and Methodology In June 2, 2013, the corpse of a woman was found floating on the Genil River on its way through the city of Granada (UTM WGS84 coordinates, X: 447102.43, Y: 4113715.98; zone 30, Northern Hemisphere). Members of the Policıa Nacional attended to the site of the discovery and reported that the identity of the victim could correspond to that of a woman with a psychiatric record, who had disappeared 17 days earlier. According to the statements from several witnesses present at the time of the disappearance, it was most likely a suicide, but they were not able to establish the exact point of entry into water. The corpse was taken to the Institute of Legal Medicine of Granada, where an autopsy was performed in accordance with current European regulations (12). The entomological evidence was collected by the coroner in charge of the autopsy according to the standards and guidelines published by the European Association for Forensic Entomology (EAFE) applicable to the terrestrial cadaveric fauna (6) as specific guidelines for aquatic fauna were not considered in that protocol. It was also registered the water temperature at the time of the corpse removal from the water (13°C). One day after the autopsy, some points of the river bottom around the discovery zone were sampled with a dipper. Regarding morphological identification, external features were observed with the aid of a dissecting microscope. After proteinase K tissue digestion, head capsules slides were mounted in DMHF (dimethylhydantoin formaldehyde resin). The specimen identifications were performed using specific taxonomic keys (13,14). To confirm the morphological identification and discriminate among related or cryptic species, total DNA was extracted from body of larvae using the Bio Sprint 15 DNA kit (Qiagenâ GmbH, Germany). Partial sequences of cytochrome oxidase subunit I (COI) were amplified by polymerase chain reaction (PCR) using the primers LCO1490 and HCO2198 (15). The PCR mix contained 2 lL DNA, 2.5 lL of the corresponding buffer, 1.5 lL MgCL2 (25 mM), 0.5 lL dNTPs mix (10 mM each), 1.25 lL of both primers (10 lM), 0.3 lL Taq DNA polymerase (5 U/lL) (Promegaâ Madison, WI, USA), and ddH2O for a total volume of 25 lL. The following PCR conditions were used in the amplifications: 94°C (4 min); 5 cycles of 94°C (45 sec), 40° (1 min), 72° (1 min); 35 cycles of 94°C (45 sec), 44° (1 min), 72° (1 min); and a final extension at 72° (10 min). The products were visualized with blue light on 0.8% agarose gels stained with SYBR Safe (Invitrogenâ Carlsbad, CA, USA) with comigrating 100 bp:1 kb ladder molecular weight markers to confirm the correct amplification. The amplified fragments were purified by ethanol precipitation prior to sequencing both strands using BigDye Terminator kits (Applied Biosystems, ABIâ Foster City, CA, USA). Products were electrophoresed on ABI 3730 genetic analyser (Applied Biosystemsâ). The forward and reverse DNA sequences obtained for each specimen were
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aligned and checked using the Sequencher software (Gene Codes Corporationâ Ann Arbor, MI, USA.) after removing primer regions. To verify the species identification, a BLAST analysis was conducted.
Results Autopsy Findings The body was in initial putrefactive conditions, with widespread green staining especially intense on the chest and neck (Fig. 1). Apart from the putrefactive dermo-epidermal detachment, a very intense maceration was noted, mainly in hands and feet. The corpse was covered by a single layer of clothing, irregularly smeared with mud compatible with the bottom of the river. No signs of external violence were observed. Given that the corpse was discovered floating and no nail detachment or evisceration was observed, the decay stage was classified as “early floating” according to Haefner’s description of submerged carcasses (16). The macroscopic internal examination was severely limited because all organs and systems were affected in varying degrees by decomposition. The weights of the most relevant organs are summarized in Table 1. The macroscopic findings on the respiratory system, stomach, and duodenum were remarkable: incipient Paltauf’s spots were observed in lungs accompanied by internal hemorrhagic suffusions evidenced when cut and intense congestion of trachea and bronchi. The respiratory airways were clean and no tracheobronchial foam fungus was observed. In the stomach and duodenum, atrophic mucosa and a water content exceeding 500 mL were easily observable.
FIG. 1––Head and neck of the corpse. Note the generalized green stain, detachment of the skin and mud smears.
TABLE 1––Weight of most relevant organs examined during the autopsy. Body area Thorax Abdomen
Organ
Weight (g)
Right lung Left lung Heart Spleen Right kidney Left kidney
405 345 275 100 125 120
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The comparative analysis of diatoms observed in closed viscera and the water recovered from the place of the corpse removal, histological findings and serological values of strontium (17,18) were consistent with a death by drowning. Entomological Findings The only entomological evidence collected during the autopsy was interposed among the long hairs of the head, in the area close to the scalp. They presented vermiform appearance, but with a distinct head capsule (eucephalic larvae) (Fig. 2A), ventrally arranged mouthparts (Fig. 2B) and thoracic appendages or prolegs along with lateral tubules in terminal abdomen (Fig. 2C), characteristic features of the Chironomidae family. A row of radially arranged grooves from mandible (Fig. 2D), typical from Chironomus larvae, was observed. Morphological analysis led to conclude that the specimens recovered from the autopsy were IV instar larvae of C. riparius. Water temperature in the moment of the corpse recovery was 13°C and during the following days later it did not change. No larvae with external morphology compatible with the previously mentioned species were captured during the river sampling near the corpse location. One of three sequences obtained (658 bases pairs) matched exactly the C. riparius sequences found in GenBank (e.g. accession numbers HM137887—coverage 100%, HM137893 and HM137888—coverage 99%) (19). The other two, which also blasted to C. riparius (in their case, with an identity of 99%), showed only one substitution with respect to the first one in the position 271. These results confirmed the presumptive morphological identification. Discussion The time of death is a major medical–legal problem faced by forensic specialists in daily practice. Immersion of a body in a liquid medium adds new difficulties to those problems already present when attempting to estimate the postmortem interval
from macroscopic or biochemical evidence. Factors such as low temperatures, currents, or pollution can introduce variations that hinder the interpretation of analytical results. Foam fungus is an important marker of vital drowning, although it is not present in all cases where this mechanism of death occurs (20). In this one, the diagnosis of that sign was negative. One possible explanation for this absence is the considerable lapse of time between drowning and the corpse discovery, which may have facilitated the washing away of the airways and mucous clearance. The study of PMSI has been classically addressed by comparing the information from the current case with casuistic repositories with known PMSI. Reh’s tables from data registered in Germany are some of the most used in our region (21,22), but their use should be undertaken with caution. The main limitations of this system based on the analogy are the differences in month/temperature with the case under study and biogeographic particularities. Correction factors should be applied depending on causal circumstances found in the biogeographic zone where the model will be implemented and endogenous factors should be included (such as weight and age) to minimize variations and errors in the model (23). Regression formulae derived from quantification of potassium from vitreous humor have been used for drowning cases (24). The main problems with this technique are the integrity of the eyeball, rarely kept because of the action of fishes and macroinvertebrates, and the erosive action of obstacles during passive drag. Another biochemical method, consisting in quantification of chlorophyll a levels, has proved very effective results in PMSI estimation in both pig carcasses and inanimate objects such as ceramic tiles (16). Despite the undeniable interest of these techniques, developmental parameters of C. riparius were actually used to calculate PMSI because they have never been tested before on a forensic case and their use could be extrapolated to similar settings. The advantages offered by the use of Chironomidae as forensic indicators of time of death are considerable (25). On one hand, it is an external indicator, so that it is not subject to the
FIG. 2––Chironomus riparius morphological details (A) whole larva; (B) mentum, ventral view; (C) ventral tubules on 11th abdominal segment, lateral view; (D) mandible, lateral view, BR: basal row.
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vicissitudes of the body that may occur as a result of postmortem scavenging or other alterations. Furthermore, the early arrival of these insects to the submerged corpse (26,27) would favor the use of the same techniques used with primary terrestrial colonizers, based on the study of development of pre-imaginal stages. Phenological studies of this species conducted in other biogeographic areas have concluded that IV instar larvae were sampled in the water from March to November, completing 4–7 generations (28). Such data have no interest to our case, as the body was found in early June, which gives us an interval with a margin of error from March to June. It is therefore necessary to use either successional data or, if Chironomidae are considered as early colonizers, ontogenic development data. Most of the Chironomidae life cycle takes place in an underwater environment. The complete metamorphosis requires from a minimum of 1 week up to several years depending on the species, water temperature, and nutrient availability among other factors (29). In the particular case of C. riparius, larvae stay inside of an U-shaped tunnel about 10 mL deep, dug into the river bottom. From the tunnel, they feed on suspended particulate organic matter that is deposited on the tunnels (30). The discovery of C. riparius in the hair of the deceased may be explained given the degradative behavior of hair keratins, biochemically similar to the river bottom. For river systems, the direct contact with the medium implies that the same type of biome has many factors that affect more directly the development and faunal diversity that in the case of terrestrial ecosystems: degree of eutrophication, oxygen, food availability, temperature water, pH, and flow speed. Because of the high environment specificity of certain species (31), these can be used not only as an indicator of PMSI, but also displacement of the body. Chironomus riparius is almost cosmopolitan, able to live in waters with a moderate to high level of contamination (32), pH between 4–8 (33), and low oxygen levels (34). All these features are compatible with the site of the discovery of the corpse. Perhaps one of the biggest difficulties that must be considered is the behavior of Chironomidae in diapause (arrested development due to unfavorable environmental conditions). Terrestrial Diptera are sensitive mainly to both temperature decrease and, below a certain value (called basal temperature or lower developmental threshold), the length increase as metamorphosis progress is arrested at any stage of development. In Chironomidae, not all instars enter into diapause; for example, it is known that C. riparius usually enters in diapause in the larval instar IV, but instar III can also enter when photoperiod and temperature conditions are suitable (35). Also, diapause disturb imaginal disks development (tissue systems which give rise to adult structures), but it does not stop the growth of the individual length, allowing to observe higher individuals than the largest individuals that developed in optimum temperatures. The maximum length observed in the sampled specimens was 15 mm, which is obviously a larger result compared with the maximum that the same species reaches when breeding under controlled temperature above the basal temperature (12.6 mm at 14°C) (36), which suggests that development was below this threshold. The absence of retrospective records of water temperature impeded more accurate estimates. If, as some studies suggest, Chironomidae larvae colonize the corpse as soon as terrestrial primary colonizers do (8,27), it can be known how long the corpse remained, at least, in the water.
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Always bear in mind that there is a period of uncertainty (and therefore not measurable) between the submersion and the arrival of the various associated species. There are many works on the development of immature stages of C. riparius as it is a classic indicator of pollution from different types of water and has been extensively studied. From previously published studies, it is known that: • If we apply Mackey’s formula (37), duration from larva I to pupa at 13°C is 27.1 days. • Goddeeris and collaborators found out in an experiment carried out at Belgium that 50% of larvae develop to IV instar at 13°C in 14 days (35). In this case, all larvae were IV instar, so 14 days would be a too conservative estimation. • In laboratory controlled conditions (36), it was recorded that, at 15°C, larvae develop to pupae in 16 days. It must be taken in consideration that environmental temperatures in this case were below 15°C, so development duration of larvae may be larger. Considering those references that set the lower (36) and upper limit (37) for Chironomidae development under specific temperature regimes, C. riparius activity on the corpse had to begin 16–27.1 days before the corpse recovery (in the refrigeration chamber, given the extremely low temperatures and the absence of water, Chironomidae do not grow), that is, between May 5–6, 2013 and May 18, 2013. If we accept the interval of disappearance as true (May 17, 2013 to June 2, 2013), submersion took place most probably in the time range when PIA and the disappearance match, that is, between May 17, 2013 and May 18, 2013. All the particularities explained above vary depending on the species, so accurate identification of specimens collected is essential to draw conclusions from real casework. DNA bar coding relies on sequence variation in short fragments of DNA to serve as a unique species identifier. Variations in the sequence of cytochrome c oxidase subunit I (COI) have proven useful for the identification of many animal taxa, including insects (38), members of the family Chironomidae (39,40), and Chironomus species (11,41). Difficulties in the taxonomy of Chironomus species are due to the existence of large complexes of cryptic species, which can be best distinguished by the banding pattern of polytene chromosomes (42). Cytological characterization, however, requires considerable expertise and is also time consuming, so molecular-based approaches, such as DNA bar coding, are being used to supplement traditional taxonomic methods of species identification. This case is a clear example that a detailed knowledge of the developmental biology of the Chironomidae can shed light on questions that otherwise might be answered only circumstantially or with higher error margins. The number of aquatic species about which we know details applicable to forensic practice are few and almost always phenological data offers too broad margins. Protocols for collecting and interpreting evidence for aquatic fauna are necessary to harmonize practices and transfer results in the context of forensic practitioners. Acknowledgments We would like to thank Annie Machordom, senior researcher and laboratory chief at the Museo Nacional de Ciencias Naturales, for providing the technical resources for the realization of molecular biology analysis.
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