William Andres Martínez Carlos Julian Idrobo Death's Insects Project Forensic Enthomology Section, Bioanthropological Lab Natural History Museum Universidad del Cauca, Popayán – Colombia E-mail: wmarti@ucauca.edu.co cjidrobo@ucauca.edu.co Keywords: ODS, Human liver, Ecological conditions, PMI, Microhabitat, Forensic En-tomology.

INTRODUCTION: The order Diptera con-tains the true flies, of which typical adults have only one pair of wings, the second pair has been modified as halteres or balancing or-ders (Smith 1986) . These hexapodous (Hexa: six, podous: legs) are holometabolous, it means that, they present a complete metamor-phosis development, showing four different stadiums: egg, larva, pupa and adult or imago. The previous ones are the mature characteris-tic dipterans. Immature states are described below. The Diptera of this study have necrophagous habits (necros: corpse, phagos: to eat) this means that they feeds on organic matter of animal origin in rotting process . The most common Diptera with necrophagous habits is the well-known Calliphoridae family or blow

flies, so this study was based on this family (Figure 1)

* This study belong to “carrion feeders Insects found on human liver in rotting process in specific micro-habitat”. In charged of Death’s Insects Project inside Bioanthropological Lab.

Ranges of Permanency of Different Observable Development Stages (ODS) of Dipterous (Arthropoda: insecta) Necrophagous in Human Liver.

© 1998 – 2000 Boletín Virtual ENLACES

SUMMARY: The forensic entomology is a discipline that uses knowledge about insects and other arthropods with le-gal purposes, it constitutes the theoretical framework of this investigation, in which 47 samplings were captured during a month (June 26 to July 26 1999) from a sample of approxi-mately 500 g. of human liver, located in a river hillside with bush and herbaceous vegetation constantly intervened (ruderal zone) to 1765 at sea level (Popayán - Colombia). 300 Dipterous were collected approximately on its different development stages. The effect of some ecological condi-tions (Tº, Hº space ubication) was the result obtained in Dipterous, besides the duration of the ODS, which are pro-posed experimentally as intervals post-mortem in similar microhabitat to the previously described.

Boletín Virtual ENLACES Edición Especial (14 de Febrero del 2000) Edición Nº 3 Vol 3, 16–25 Articulo No. bve04140200

Figure 1 Calliphoridae adult (Illustrated by Roxanna Perafán)

ODS: The observable development stages (ODS) as it has been denominated the obser-vations that were carried out in studied dip-terans, weredefined in this way because it facilitated their field identification, the ODS are: Eggs are characterized to possess a small size, approximately of some 1,6mm of length, they are of cream white colour and present a central band, they are found in masses of hundreds in holes, caverns of tissue and un-der it (Figure 2). This description is carried out based on Calliphorid eggs that were ob-served and collected.

Larvae are grubs or maggots. Three instars of larvae can be identified (they were not dif-fered in this study), they are called at way since eggs hatch until prepupal state; they are of a cream yellow colour and have a con-spicuous dark stain in the internal part of their stomach, due to the rotted tissue in their bowels. They are vermiform, acephalous, without wings and their size oscillates be-tween 1mm and 20mm (Figure 3).

The pupa is the dipterans latency state, these are found inside a dark brown shell known as puparium and measures 10mm approxi-mately, characterized to possess traversal bands (Figure 4).

The last development state corresponds to the adults or imagos previously described. FORENSIC ENTOMOLOGY: For more than a century, beginning with Bergeret

W. A. MARTINEZ AND C. J. IDROBO

© 1998 – 2000 Boletín Virtual ENLACES

10 mm

Figure 2 Eggs over human liver fragment, each square lengthens 5mm per side (Photograph by Astrid Perafán)

Figure 3 Calliphorid larvae (Photographied and illus-trated by Roxanna Perafán).

Figure 4 Calliphoridae puparium (Illustrated by Rox-anna Perafán)

RANGES OF PERMANENCY OF DIFFERENT STAGES (ODS)

works in 1855, followed by the classic work of Jean Pierre Megnin (1894) (Haskell et al, 1996), necrophagous insects knowledge has been used with medical-legal proposals, be-ing a useful tool to estimate post-mortem in-tervals (PMI). PMI′s estimation is carried out based on pre-sent arthropods specia and development stages that can be seen on them (Oliva, 1997) The forensic entomology is a discipline that is giving it first steps in South America. Pro-fessor Oliva (1997) from Forensic Entomol-ogy Laboratory of Natural History Museum ‘Bernardino Rivadavia’ in Buenos Aires Ar-gentina, mentions that first studies about this area were carried out by Freire (1914), Pes-soa (1935), Pessoa and Lane (1941) as iso-lated speciality applications. In Colombia, forensic entomology, only can register works carried out in canids at “Universidad del Valle” by Olaya (1998) and at “Universidad de Antioquia” with studies carried out by Wolff (with unknown publica-tions). With the absence and the necessity of an in-vestigation on this area, in 1997 the Death’s Insects Project was born as the forensic en-tomology section inside the Bioanthropologi-cal Lab of the Natural History Museum of the “Universidad del Cauca”. From then on it has come a conceptualising time on this dis-cipline. The main objective of this study was to know the duration time of each one of the ODS of dipterans that feed on human liver fragments in rotting process in a specific microhábitat. The results presented here conform the com-parison target for future proofs, which will make us able to affirm ODS´s applicability to estimate post-mortem intervals.

STUDY AREA: The study was carried out in the Bioterium of the health scinces campus of the “Universidad del Cauca”, in a ruderal zone located on a river hill side besides the Molino river (Popayán-Colombia) at 1765 meters over sea level (Figure 5). The initial characteristics of the place were:

Soil conformation of sampling zone is com-posed by a superficial coat of dry leaves in degradation, followed by a thick coat of frank - loamy material with sand in high pro-portion, probably deposited there because the grow of the river, that is a few meters near.

© 1998 – 2000 Boletín Virtual ENLACES

Figure 5 Study Zone (Photograph by Roxanna Perafán)

Topographically the place is plain it , inside Popayán´s urban area, and it is located on Pu-benza’s Valley. For soil chemical conditions it is known that Cauca’s region is character-ized to have a sour type pH. The environmental temperature at twelve o’clock; time of maximum dipteral activity, had an average of 22.78°C, with a standard deviation similar to 2,96ºC, a variability of 13,01% indicating that it is statistically vari-able. The registered minimum temperature was 16°C, this fact was taken after a strong rainfall, coinciding with a relative humidity of 86% being the highest registered at that hour; the maximum temperature (29°C) coin-cides with a relative humidity quite far from the average (32%) and for that is possible to infer, that the temperature in this microhabi-tat sustains a practically direct relationship with humidity. The soil temperature had an average of 19,70°C, a standard deviation similar to 2,30ºC, indicating a variability coefficient of 11,67%, as considering statistically variable. The minimum temperature similar to 18°C and maximum of 28.8°C also coincides with the lowest relative humidity (32%). When comparing the two previous variables it is re-markable the relative stability of the floor in relation with the atmosphere for thermal variations, it can be inferred that it is because in the first system (soil), stored water concen-tration is bigger than in the air, which allows its stability (Figure 6). Environmental relative humidity has been calculated in an average similar to 59,37%H, a standard deviation similar to 14,87%H, with a variability coefficient of 25,05%, be-ing a system statistically very variable, the minimum similar to 22% and the maximum similar to 86% previously related with the other two variables. (Figure 6)

Tree, bush and herbaceous vegetation was found in and surrounding the sampling zone, represented by plants belonging to the follow families: Euphorbiaceae (Phyllanthus salvi-ifolius), Rutaceae, Balsaminaceae abundant in underwoods, Tropaeolaceae inside ‘Pudridero’ (see below, On Materials and Methods), Amaryllidaceae surrounding sam-pling zone, and others who were less abun-dant, almost all the found flora is typical from constantly intervened zones (Gentry 1993). Another kind of fauna was observed: insects from the follow orders: Diptera, mainly he-matophagous, Lepidoptera, Collembola, Hy-menoptera, Orthoptera, Dermaptera, Odonata and Hemipterous; In what concerns Arthro-pods in general, an enough presence of Aracnea and normal presence of typical soil invertebrates. As for vertebrates, some birds (Corvidae) surrounding sampling zone and traces of Canids are observed, in the prox-imities of the river, rats and opossums. MATERIALS AND METHODS: The site was built inside study area that allows the en-trance and exit of the dipterans and other in-sects, but protecting the bait from vertebrates as dogs and rats. This place has been denomi-

(Continúa en la página 5)

© 1998 – 2000 Boletín Virtual ENLACES

W. A. MARTINEZ AND C. J. IDROBO

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Time (days)

Tem

p(°C

) y %

Hum

T°ambiente T°sustrato H°ambiente

Figure 6 Temperature and relative humidity behav-iour in time function.

nated as ‘Pudridero’. Pudridero is a cubical concrete construction (1m x 0.5m x 1m) whose bottom is of same soil of the surroundings like graphics shows (Figure 7).

BAIT: As bait was employed a human liver of 500g approximately, which was frag-mented to facilitate the internal observations. Human liver is used, because provides an ap-propriate medium for ovoposition and devel-opment dipterans to study (Figure 7). The liv-ers for the study are donated by the National Institute of Legal Medicine, Sector Cauca; under legal and conscience ethics norms of human being respect and their conception of death with their social implications.

The employment of human liver as bait has caused controversy, because is of common knowledge that pig liver -for example-, is very similar to human liver or its rare to find a corpse with liver exposed or find visors alone, but in bibliographical revisions (Gerber 1993, Goff 1997, Haskell 1996, Ke-iper 1997 knicht 1994, Krogman & Iscan 1986, Oliva 1997 Reverte 1991, Rodriguez 1994, Sadler 1997, Smith 1986, Vargas 1991), were not works where was used this as bait and is unknown if rotting processes are really the same, on the legally hand, it is only allowed to use denominated viscerec-tomy residuals for investigation, like bowels and other organs that are in the thoracic and abdominal cavity; also the original purpose of thid investigation was to work with human tissue. MONITORING AND SAMPLE’S TAKING: Were carried out 47 monitorings in 30 days: 12 in one hour intervals, four every three hours, two every six hours, later an interval of 18 hours that allowed that following 28 monitoring were every 24 hours to be made at 1200h (Table: 1) in this way we could ob-serve all ODS of dipterans who visit the bait. To develop this sampling program we based on Smith (1987); Olive (1997) and other authors that documented some d Time

© 1998 – 2000 Boletín Virtual ENLACES

RANGES OF PERMANENCY OF DIFFERENT STAGES (ODS)

Figure 7 Pudridero different profiles (Photograph by Roxanna Perafán; Illustrations by Authors)

Figure 8 Different views from bait (Photographs by Roxanna Perafán (Left) and Astrid Perafán (Right).

© 1998 – 2000 Boletín Virtual ENLACES

W. A. MARTINEZ AND C. J. IDROBO

Sample Number

d Time In-terval

Accumulate Interval

Local Time

0 1 0h 0h 0600h 1 1 1h 1h 0700h 2 1 1h 2h 0800h 3 1 1h 3h 0900h 4 1 1h 4h 1000h 5 1 1h 5h 1100h 6 1 1h 6h 1200h 7 1 1h 7h 1300h 8 1 1h 8h 1400h 9 1 1h 9h 1500h

10 1 1h 10h 1600h 11 1 1h 11h 1700h 12 1 1h 12h 1800h 13 1 3h 15h 2100h 14 1 3h 18h 2400h 15 1 3h 21h 0300h 16 1 3h 24h 0600h 17 2 6h 30h 1200h 18 2 6h 36h 1800h 19 2 18h 54h 1200h 20 3 24h 78h 1200h 21 4 24h 102 h 1200h 22 5 24h 126 h 1200h 23 6 24h 150 h 1200h 24 7 24h 174 h 1200h 25 8 24h 198 h 1200h 26 9 24h 222 h 1200h 27 10 24h 246 h 1200h 28 11 24h 270 h 1200h 29 12 24h 294 h 1200h 30 13 24h 318 h 1200h 31 14 24h 342 h 1200h 32 15 24h 366 h 1200h 33 16 24h 390 h 1200h 34 17 24h 414 h 1200h 35 18 24h 438 h 1200h 36 19 24h 462 h 1200h 37 20 24h 486 h 1200h 38 21 24h 510 h 1200h 39 22 24h 534 h 1200h 40 23 24h 558 h 1200h 41 24 24h 582 h 1200h 42 25 24h 606 h 1200h 43 26 24h 630 h 1200h 44 27 24h 654 h 1200h 45 28 24h 678 h 1200h 46 29 24h 702 h 1200h 47 30 24h 726 h 1200h

biological cycles of some necrophagous dip-terans. Sample Number PROCEDING IN EACH MINITORING:Registered data of ambient, bait and soil tem-perature, environmental relative humidity, ODS, collected specimens, atmospheric con-dition and photographic registration was gathered to later analysis. Collected insects was found in decomposition tissue, surroundings and flying over. Capture method was manual (Moron and Teron, 1900), with jama for mature flies and pincers or forceps for immature stages (Figure 9). The adults were sacrificed inside a xilol lethal chamber, then were preserved in ethylic alco-hol at 70% in which were added approxi-mately 4-5 drops of glycerine per liter, this mixture favors to conserves specimens tex-ture.

Some alive animals (eggs, larvae and pupas) were kept in an aired and humid recipient with liver fragments in decomposition to fin-ished their development at lab, facilitating their identification. SAMPLING AREAS: The sampling areas were defined like intern and external, The first ones refer to nine quadrants inside

Table 1: Sampling Program

Figure 9 Sampling (Photograph by Roxanna Perafán)

adults emerging, finally ware seen only Cal-liphorids;. This behaviour is presented in the table 2 and it is summarized in the table2.

O.D.S. Ranges of permanency (in hours) H L P A

3-8

9-24 30-102

126 150-294

318-678 702

DISCUSSION: ODS space location was me-diated by microhabitat temperature and hu-midity taken place by sun exposure; where was shade, temperature kept lower and rela-tive humidity high and stable, over this places were observed bigger ODS incidence and among shadier was more Calliphorids imma-ture states, indicating a contagious space lo-cation (Ravinobich 1978) with likeness for already mentioned places (Figure 11).

Is noticed in graph at 22nd sampling, eggs are unobserved; but adults continue visit bait, this is because adults go to bait carry out by dif-ferent activities as feeding of remainders liq-

this; since 30h until 678h larvae were ob-served also in their majority belonging to Calliphorid, to 150h-726h dark brown pupas were observed, some of this were collected to finished their development at Lab until saw adults emerging, finally was seen only Cal-liphorids;. This behaviour is presented in the chart and it is summarized in the table2.

Pudridero and eight constitute the external ones limited by the maximum distance of lar-vae dispersing. (Figure 10).

RESULTS: In total were captured around 300 insects in which almost of those (80%) was dipteral, mainly belonging to Calliphorid fam-ily. The collected specimens shortage is due with study objectives accordance, was un-needed a great specimens number , since it was not sought to make a populational analysis, simply to a taxonomic insects describing and their developing in time function, also planed in this way to not intervening too much among the tissue in putrefaction process. Because the biggest incidence of dipterans cor-responded to Calliphoridae family, ODS analy-sis was based alone on these. The maxim distances traveled by larvae was 2m, regarding Pudridero walls. Inside microhabitat was register ODS and other ecological differences like previous graphics shows. After data gathering with ODS regard on bait and their proximities were located in time evi-dencing that: of 9h-102h eggs were observed belonging to Calliphorid observed flying over, the eggs were found in liver holes and below

Internal

External

Figure 11 Inmatures states distribution in icrohabitat

Figure 10 Pudridero and sampling zone

Table 3 ODS summary.

© 1998 – 2000 Boletín Virtual ENLACES

RANGES OF PERMANENCY OF DIFFERENT STAGES (ODS)

Dia

# M

EDO

H L P A

EDO

Intervalo

0. 0h 1. 2. 2h

3. A 3h

4. 5.

1 6.

7.

8. 8h

9. H y A 9h

10.

11. 12.

13.

14.

15.

2 16. 24h 17. H,L y

A 30h

18. 3 19.

4 20.

5 21. 102h

6 22. L Y A 126h

7 23. L,P y A

150h

8 24. 9 25.

10 26.

11 27.

12 28. 13 29. 294h

14 30. L y P 318h

15 31.

16 32.

17 33. 18 34.

18 35.

20 36.

21 37.

22 38.

23 39.

24 40.

25 41. 26 42.

27 43.

28 44.

29 45. 678h 30 46. P 702h

Table 2 ODS permanency ranges. H eggs. , L Lar-vae, P puparium, A Adult

W. A. MARTINEZ AND C. J. IDROBO

© 1998 – 2000 Boletín Virtual ENLACES

uids or because they belong to other species, in these case ovoposition was not observed. It is also remarkable that the first adults don't take a lot in to going to the bait (3h) agreeing with several authors. Larvae and pupas appearance coincide with a logical chronological development, because since first eggs are observed until first larvae appearing a time interval (21h) chord with fly normal development, the same thing happens to pupas regarding larvae which begins to ob-serve 120 hours after first larvae are observed (Haskell 1996). ODS OPEPERATION TO ESTIMATE PMI WORKS IN THE FOLLOWING WAY: If a corpse is found in a microhabitat with similar meteorological and ecological characteristic with study place ones, collected Calliphorids in larval and pupal states, could be assume a PMI range in summary table accordance of 318-678h, if one observes attentively can see that ranges are relatively wide; but, it can decrease remitting us to some peculiarities like larvae and pupal maturity stage. If only pupas has found a 702 h PMI is considered. ODS applicability as PMI estimates is based in along permanency time of each one ODS is different referring to individuals that share developments and similar ecology. Also, it should be kept in mind the exact place where are collected or observe each one of ODS and the previous meteorological re-ports to corpse found moment; according to the graph, in each place due to temperature differences and microhabitat location slows or accelerates dipterans development. Is im-portant to collect dipteran in all the places where thay are, to be able to carry out an esti-mate by the proposed pattern. To be the bait only composed by liver and when area is being constantly intervened by

investigators samplings, an error range is generated to PMI estimating with this model, making a comparison with a hypothetical real case where corpse remains keeps without hu-man intervention until it is found and ana-lysed. Human liver represents an ideal environment for necrophagous dipterans development cy-cles observation, can be rejected the intere-specific intrinsic variable inside a research with human different species. Is observed that ecological variables are a point to keep in mind at ODS evaluating to estimate shorts PMI (until one month), when applying this information to estimate a trust-worthy PMI. The method outlined at the same time of being punctual to microhabitat specificity conditions is a base to extrapolate data for later investigations, with building purpose of a flexible and equally faithful method. REFERENCES GERBERT, V. 1993. PRATICE HOMI-CIDE INVESTIGATION, TACTICS, PRO-CEDURES, AND FORENSIC TECH-NIQUES. Second Edition. Boca Raton, Ann Arbor, London, Tokyo. GENTRY, A.H. 1996. AFIELD GUIDE TO THE FAMILIES AND GENERA OF WOODY PLANTS OF NORTHWETS SOUTH AMERICA (COLOMBIA, ECUA-DOR, PERU). The university of Chicago press. GOFF, M. Lee and WIN, Bani H. 1997. ES-TIMATION OF POSTMORTEN INTER-VAL BASED ON COLONY DEVELOP-MENT TIME ANAPLOLEPSIS LONGIPES (HYMEMOPTERA: FORMICIDAE). J Fo-rensic Sci; 42(6). 1176-1179.

© 1998 – 2000 Boletín Virtual ENLACES

RANGES OF PERMANENCY OF DIFFERENT STAGES (ODS)

HASKELL, N.H.; HALL, R.D.; CER-VENKA, V.J.; CLARK, M.A. 1996. On the Body: Insects´ Life Stage Presence and their Postmortem Artifacts en: FORENSIC TA-PHONOMY, THE POSTMORTEM FATE OF HUMAN REMAINS. 415 – 448. KEIPER JB, CHAPMAN EG, FOOTE BA. 1997. Midge larvae (Diptera: Chironomidae) as indicators of Postmortem Interval of car-casses in a Wooland Stream: a Preliminary Report en J FORENSIC SCI; 42(6): 1074 – 1079. KNICHT, Bernard. 1994. Otras formas para calcular el tiempo de muerte en MEDICINA FORENSE. Edit, El manual moderno, S.A. De C.V. México, D.F. P 40. KNIGHT, B. 1994. MEDICINA FORENSE DE SIMPSON. MANUAL MODERNO. México D.F, Santa Fe de Bogotá. KROGMAN & ISCAN 1986. THE HU-MAN SKELETON IN FORENSIC MEDI-CINE. Thomas publisher. MORON & TERON. 1988. ENTO-MOLOGÍA PRACTICA. Ediciones del min-isterio de Agricultura México D.F. OLIVA, A. 1997. Insectos de interés forense de buenos aires (Argentina). Primera lista ilustrada y datos binómicos en: REVISTA DEL MUSEO ARGENTINO DE CIENCIAS NATURALES “BERNARDO RIVADA-VIA” E INSTITUTO NACIONAL DE IN-VESTIGACIONES DE LA CIENCIA; Tomo VII N°2. Buenos Aires. RAVINOBICH, J.E. 1997. ECOLOGÍA DE POBLACIONES ANIMALES. Programa Re-gional de Desarrollo Científico y Tec-nológico. Departamento de Asuntos Científi-cos. Secretaria General de la Organización de Estados Americanos. Washington, D.C.

REVERTE, J.M. 1991. Entomología Forense en ANTROPOLOGÍA FORENSE. 404 – 461. RODRIGUEZ, J.V. 1994. INTRODUC-CIÓN A LA ANTROPOLOGÍA FORENSE. Universidad INCCA. Colombia. SADLER, D.W. Et al. 1997. Barbiturates and analgesics in Calliphora vicina Larvae en: J. FORENSIC SCI; 42 (3): 481-485. SMITH, K.G. 1986. A MANUAL OF FO-RENSIC ENTOMOLOGY. British Museum (Natural History). Cmstock Publishing Asso-ciates. Cornell University Press, Ithaca, New York. Oxford. VARGAS, E. 1991.MEDICINA FORENSE Y DEONTOLOGÍA MEDICA. Editorial Trillas. México. Acknowledgments Humberto Granados for technical, site, taxo-nomical and a very large contributions list, Esther Estrada for bait consecution, Federico Melendez for traduction contribution, Mark Benecke for final revision. Ernesto León Rodríguez, Carlos David Rodríguez, Liliana Astaiza, Astrid Perafán, Willy Montoya, Rosanna Perafán and Victor Lozano, for helping us taking the samples, methodologi-cal discussions, Pudridero´s construction and their support, To all we are greatful.

W. A. MARTINEZ AND C. J. IDROBO

© 1998 – 2000 Boletín Virtual ENLACES