Forensic Use of the Piracatinga Fish

“Callophysius macropterus” can be used to Locate and Identify Human Remains Retrieved From the Amazon River

The Amazon region is inhabited by several species of fish such as Piranhas, Candirus, and small catfish all of which are known to attack people. These fish inhabit mainly the muddy shoals of the Amazon River (1–3). The medical importance of these fish is related more to their scavenging lifestyle than to their contribution to human health (2). Piracatinga are necrophagous, and dead bodies that have sunk to the river bottom are quickly devoured (4). Piracatinga are therefore known in the region as “black water vultures.” They breed between January and February and grow during two periods of the year—one during the river flood (in January) and the other during the river ebb (in August) (5,6). They can reach 45 cm in length as they scavenge the bottom of the shoals that they inhabit (7,8). Examples of Piracatinga are shown in Fig. 1 (a, b). Aside from their forensic importance, Piracatinga are caught in large quantities to supply the market of some Latin American countries where they are considered a delicacy. To catch Piracatinga, fishermen use dolphin flesh (Inia geoffrensis and Sotalia fluviatilis) as bait, causing an ecological imbalance of the fauna in their habitat (9–12), and threatening the two dolphin species with extinction.

Although there is no clear evidence in the literature of Piracatinga attacks on live humans, anecdotal reports of Fire Department divers from small cities in the rural region along the Amazon point to Piracatinga as highly aggressive in the way that they dismember the corpses of drowning victims trapped in sunken wrecks of boats along the Amazon River bottom. Most often, the bodies of drowning victims in the Amazon are difficult to locate owing to the vastness of the river system and turbidity of its waters, or, by being devoured by fish. Families that lose one of their members on the river suffer intense psychological stress (13). According to Brazilian law, a death certificate cannot be issued until 5 years after a person has disappeared, even if the drowning was witnessed. Therefore, finding some way to acknowledge the death of a drowned person can bring significant comfort to the victim’s family. In this paper, we report two cases in which Piracatinga were instrumental in finding and subsequently identifying human remains from the Amazon River. Materials and Methods Location of Remains Following disappearance of the subject, the families involved contacted the Fire Department of the City of Santar
em. The Fire Department searched the river in the vicinity of the victim’s disappearance for signs of Piracatinga. Once Piracatinga were caught, and human remains identified in the digestive tract, the Fire Department was activated to dive and to try to recover the remains.

Materials and Methods

Location of Remains

Following disappearance of the subject, the families involved contacted the Fire Department of the City of Santar
em. The Fire Department searched the river in the vicinity of the victim’s disappearance for signs of Piracatinga. Once Piracatinga were caught, and human remains identified in the digestive tract, the Fire Department was activated to dive and to try to recover the remains.

Fishing for Piracatinga

To catch Piracatinga, chicken skin pieces were used to bait hooks. Within 5 min after lines were cast, Piracatinga began to bite, creating a feeding frenzy, examples of which are shown in Fig. 1 (c, d). The site of the individual’s disappearance was noted, and teams of experienced fishermen were requested to report changes in the water surface made by the Piracatinga (Fig. 2).

Analysis of Collected Material

Analysis of remains from the Piracatinga intestinal tracts began at the site where they were captured. The intestinal tract contents were evaluated macroscopically. In both cases presented here, there were pieces of skin and hairs with human characteristics. Samples of Case 1 remains were sent for DNA analysis. Case 2 remains were discarded as soon as the body was found by the Fire Department. mtDNA was analyzed based on the method described by Wilson et al. (14), and the mtDNA sequence determined by Anderson et al. (15). Samples of mtDNA analysis were extracted by standard methods, and the two hypervariable (HV) regions, HVI and HVII, were amplified by PCR to obtain sufficient DNA for sequence analysis.

Results

Case 1

A 56-year-old male dived off a boat from a height of approximately 4 m while sailing on the Amazon River. He hit the water with high impact, lost consciousness and drowned. The crew of the boat saw his body floating motionless and face down. He sank shortly thereafter leaving no time for attempted rescue, and the search for the body began immediately after the drowning victim disappeared. Despite an intense effort over the following 5 days of searching, the remains of Case 1 were not recovered. In Case 1, the body was not recovered but the location of the disappearance was noted by witnesses to the accident and served as a basis for the location where to fish for Piracatinga. That, together with disruption of the river surface by Piracatinga, served as a guide where to locate fish that may have ingested some of the remains. Material that was removed from the digestive tract of the Piracatinga was sent for DNA analysis. The sequences of the primer pairs used for DNA amplification by PCR were:

L15996 (50-CTCCACCATTAGCACCCAAAGC-30) and H16391 (50-GAGGATGGTGGTCAAGGGAC-30) for HVI and L48 (50-CTCACGGGAGCTCTCCATGC-30) and H408 (50-TGTTAAAAGTGCATACCGCCA-30) for HVII.

Amplification was for 36 cycles, and the resultant amplimers were gel-purified and sequenced by standard techniques. DNA from skin and blood samples of one of the sisters of the victim was analyzed with the conclusion that the two sets of samples were derived from offspring of the same mother.

Nine Piracatinga, collected at the location where the victim was last seen, were caught in two batches—the first batch (consisting of six Piracatinga) was caught within 48 h of the accident; the second batch (consisting of three Piracatinga) was caught between 48 and 72 h after the accident. Of the nine fish caught, five contained material resembling human tissue in their digestive tract. In samples obtained from three fish, it was possible to identify macroscopically, tissue whose appearance was consistent with human skin, including the presence of hair. The other two Piracatinga digestive tract contents contained material that was suggestive of adipose tissue in an advanced stage of digestion. The remaining fish caught were discarded.

Fragments of the remains were preserved in formaldehyde. The preserved material was registered with the Civil Police section of Santar
em City, with detailed reports of witnesses testifying how the material was collected. After the judicial proceedings, the material was analyzed by conventional histopathological methods to ensure that it consisted of human tissue. Microscopic findings revealed apocrine and eccrine glands, hair follicles, and other characteristics that confirmed the identification as human skin. The epidermis showed signs of exposure to digestive enzymes, likely from Piracatinga. Thereafter, the material was sent for mitochondrial DNA analysis, carried out in the National Forensic Institute of the Federal Police in the City of Brasilia (Instituto Nacional de Criminalística da Polícia Federal de Brasília), as described in the Materials and Methods.

Case 2

A 51-year-old male resident of the right bank of the Amazon River left home to go to a social gathering 200 meters distant from his home, but he failed to return. After a disappearance of 24 h his family began a search of his home. Some of his relatives fished for Piracatinga on the banks of the river between the locations of the victim’s home and the gathering. The relatives noticed movement on the river surface of fish shoals that imparted a characteristic pattern, and raised the possibility that the victim drowned or that might have been murdered; locals said that he might have had an altercation at the home gathering the night he disappeared. The remains of Case 2 found 48 h postmortem with partly eaten chest cavity and left arm are shown in Fig. 3.

In Case 2, Piracatinga had devoured the entire contents of the abdomen, intrathoracic organs, and almost all of the musculature and subcutaneous tissue in <48 h. After the body was located, a more detailed examination of the site near the riverbank where the victim disappeared led to the discovery of fresh drops of blood on a sandy stretch. The blood trail led to the river bank where the body was located and was not analyzed further. A partially decomposed body was recovered, located by the Fire Department precisely where the Piracatinga were captured. In this case, it was considered unnecessary to send the human remains for DNA analysis, as the family identified the body. The tentative identification of the remains was confirmed on the basis of money that was found in the victim’s pocket, jewelry that was still around his neck, hair color and texture, a gold tooth, clothing remnants, and a wallet containing personal documents of the deceased. Murder could not be ruled out by the Institute of Legal Medicine due to the considerable loss of tissue and organs devoured by the Piracatinga.

Discussion

Fatal drowning is the world’s largest cause of death among young people ages 5–14 years—with Brazil having the third highest number of such cases in the world (16,17). In the Amazon region, deaths from external causes are the main cause of death in some cities (18). The region has the highest incidence of accidental drowning in Brazil, not counting shipwrecks, where many people die (19). Many victims of drowning in the Amazon region, especially on the river, are not found—either because of the vastness of the river or by being devoured by carnivorous or scavenging fish such as Piracatinga. Moreover, data from the medical literature on drowning in the Amazon are scarce (17).

In this paper, we illustrate two cases where the victims of drowning could be identified owing to the characteristic disruption of the river surface made by the Piracatinga when they feed. Characteristic changes seen on the surface of the river when Piracatinga are feeding consist of concentric circular waves with a major disruption at the center and bubbles that only Piracatinga make. Piracatinga nasal barbels penetrating the surface can also be seen. In contrast, Piranha usually do not disrupt the water surface when they are feeding. Moreover, changes in the river surface, if any, due to feeding Piranha, are not accompanied by formation of bubbles. Although well known by the locals, the possibility of such identification as described in the present report is, for the best of our knowledge, the first in the medical literature.

The legal and psychological problems arising from the loss of family members, as described above, are considerable. Unfortunately, Brazilian law stipulates that an individual can be considered legally dead, without identification of remains only after 5 years following disappearance (20).

Elsewhere, out of the water, forensic science uses necrophagous insects to detect the location where an individual died. Several species of necrophagous insects are attracted by the odor of a decomposing body (21). Piracatinga are not unique in their usefulness as a source of human remains following a fatal incident in the Amazon. Other samples of human remains have been obtained from fly larvae (de Lourdes et al. (22), Campobasso et al. (23)) and sharks, Byard et al.(24). The significance of Piracatinga is based on the accelerating economic development of the Amazon and the increasing need to identify the remains that result from the fatal incidents that follow.

Unfortunately, the use of insects is limited to merely locating remains. Anecdotal reports from local and other riverine fishermen have noted that Piracatinga tend to congregate in places where humans or animals have disappeared, and that they often show human remains in their digestive tract. We are unaware of scientific reports describing the use of Amazonian fish as bioindicators of the presence of human remains resulting from fatal drowning. More broadly, these studies open a discussion about the value of anecdotal information to advance medicine, health, and the interrelation with the environment in the Amazon.

Even considering the vastness of the Amazon basin, fishing for Piracatinga in places where there is the presence of dead animals or humans becomes relatively easy. The Piracatinga are extremely voracious fish; they feed on shoals and have territorial habits, so that they are known as “water vultures” (6,18). In the cases presented here, collecting the specimens of Piracatinga required, on average, about 2 h per day.

Careful examination of the digestive tract of these fish can be carried out in situ. Their dental arch and jaw are strong and usually tear off pieces of skin of considerable size (25,26). In Case 1, the presence of scalp hairs was easily recognized by macroscopic examination. In Case 2, the fish localized the presence of the body in an improbable location. It was expected that the victim’s body would be located at a more distant site because of the river current. By the time the fish were caught, the family was unsure what had happened. After the Piracatinga were examined, the possibility of death became evident. Unfortunately, it was not possible to determine the cause of the death. As the remains could not float due to the significant loss of tissue, the presence of Piracatinga was fundamental to locating the body. In Case 1, the body was not found, possibly due to the greater depth of the river and irregularity of the riverbed at the investigation site.

The histopathology examination, prior to DNA examination, proved useful in Case 1 as it demonstrated histological characteristics of human skin and alterations in the epidermis due to the probable action of digestive enzymes of the fish. Although the sizes of the skin samples collected from the Piracatinga were of reasonable size for a conventional DNA study, unfortunately the use of formaldehyde to preserve the material impaired its use. Ideally, the collected samples should be preserved in 70% ethanol (27) at the time they are collected. Despite this, the analysis of mitochondrial DNA was possible and it has the advantage of greater sensitivity because a smaller amount of material is needed to define if an individual is part of the same maternal lineage. Evidence that the fragments of skin and hairs removed from the interior of the Piracatinga belonged to the individual (Case 1) helped the judiciary to issue the death certificate in <2 years.

In summary, Piracatinga serve as a useful tool for locating the remains of drowning victims in the Amazon. When combined with comparative analysis of DNA samples from relatives of the deceased, the method can serve as a powerful forensic tool both to locate victims’ remains and to determine victims’ identity.

 

Acknowledgments

The author acknowledges the assistance of Bernard Weisblum and Marcos Colón in preparation of the manuscript.

Department of Neurosurgery, University of Par
a-State and Hospital Regional do Baixo Amazonas, Santarém, Pará- Brazil.

References
1. Bauer LI. Candiru–a little fish with bad habits: need travel health professionals worry? A review J Travel Med 2013;20(2):119–24.

2. Haddad V Jr. Animais aquáticos de importância médica no Brasil [Aquatic animals of medical importance in Brazil]. Rev Soc Bras Med Trop 2003;36(5):591–7.

3. Sazima I, Zuanon J, Haddad V Jr. Puncture wounds by driftwood catfish during bucket baths: local habits of riverside people and fish natural history in the Amazon. Wilderness Environ Med 2005;16(4):204–8.

4. Ramirez-Gil H, Feldberg E, Almeida-Val VMF, Val A. Karyological, biochemical, and physiological aspects of Callophysus macropterus (Siluriformes, Pimelodidae) from the Solim~oes and Negro Rivers (central Amazon). Braz J Med Biol Res 1998;31(11):1449–58.

5. Perez A, Fabré NN. Seleção das estruturas calcificadas para a determinação da idade da Piracatinga Calophysus macropterus Lichtenstein (Siluriformes: Pimelodidae) na Amaz^onia Central [Selection of calcificated structure for aging of the piracatinga, Calophysus macropterus Lichtenstein (Siluriformes: Pimelodidae) in the Central Amazon, Brazil]. Brasil Acta Amaz 2003;33(3):499–514.

6. Perez A, Fabré NN. Seasonal growth and life history of the catfish Calophysus macropterus (Lichtenstein, 1819) (Siluriformes: Pimelodidae) from the Amazon floodplain. J Appl Ichthyol 2009;25(3):343–9.

7. Estupinãn G, Marmontel M, Queiroz HLD, Souza PRE, Valsecchi J, Batista GDS, et al. A pesca da Piracatinga (Calophysus macropterus) na Reserva de Desenvolvimento Sustentável Mamiraua [Piracatinga fish (Calophysus macropterus) in the Mamirau
a Sustainable Development Reserve]. M.r.d.C.n.e.T.Technical report. Tefe, Amazonas, Brazil: Institute of Sustainable Development Mamirauá, 2003.

8. Trujillo F, Gomez C, Portocarrero M, Diazgranados MC. Conservacion de la diversidad biologica y cultural [Conservation of biological diversity and cultural]. In: Ruiz SL, Sanchez E, Tabares E, Prieto-C A, Arias-G JC, Gomez R, Castellanos D, Garcıa P, Rodrıguez L, editors. Diversidad biologica y cultural del sur de la Amazonia colombiana [Biological and cultural diversity of the south of the Columbian Amazon]. Bogota, Colombia: Corpoamazonia, Instituto Humboldt, Instituto Sin-chi, UAESPNN, 2007;440.

9. Batista SV, Barbosa WB. Descarte de peixes na pesca commercial em Tefé, médio Solimões, Amazônia Central [Discarded fish from commercial fisheries in the middle Solimões river, Central Amazon region]. Acta Sci Biol Maringá 2008;30(1):97–105.

10. Iriarte V, Marmontel M. River Dolphin (Inia geoffrensis, Sotalia fluviatilis) mortality events attributed to artisanal fisheries in the western Brazilian Amazon. Aquatic Mammals 2013;39(2):116–24.

11. Mintzer VJ, Martin AR, Silva VMF, Barbour AB, Lorenzen K, Frazer TK. Effect of illegal harvest on apparent survival of Amazon River dolphins (Inia geoffrensis). Biol Conserv 2013;158:280–6.

12. Sá Alves LCP, Zappes CA, Andriolo A. Conflicts between river dolphins (Cetacea: Odontoceti) and fisheries in the Central Amazon: a path toward tragedy? Zoologia 2012;29(5):420–9.

13. Lenferink LI, Wessel I, de Keijser J, Boelen PA. Cognitive behavioural therapy for psychopathology in relatives of missing persons: study protocol for a pilot randomised controlled trial. Pilot Feasibility Stud 2016;2:19.

14. Wilson MR, DiZinno JA, Polanskey D, Replogle J, Budowle B. Validation of mitochondrial DNA sequencing for forensic casework analysis. Int J Legal Med 1995;108(2):68–74.

15. Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, et al. Sequence and organization of the human mitochondrial genome. Nature 1981;290(5806):457–65.

16. WHO. Injuries and violence prevention: non-communicable diseases and mental health: fact sheet on drowning. Geneva, Switzerland: World Health Organization (WHO), 2003; http://www.who.int/violence_injury_prevention/ other injury/drowning/en/index.html.

17. Szpilman D, Bierens J, Handley AJ, Orlowski JP. Drowning. N Engl J Med 2012;366(22):2102–10. 4 JOURNAL OF FORENSIC SCIENCES

18. Vieira G, Basano SA, Camargo LMA. Transition of the morbidity and mortality profile in a municipality in the interior of the Brazilian Amazon. Rev Soc Bras Med Trop 2016;49(4):411–7.

19. Http://Datasus.gov.br/cgi/tabcgi.exe?sim/cnv/ext10uf.def.

20. Cartilha Jurídica do Luto: orientações práticas e jurídicas aos familiares / Escola de Direito do Rio de Janeiro da Fundação Getúlio Vargas [Legal guide to mourning: practical and legal guidance to family members/Getúlio Vargas Foundation Law School of Rio de Janiero]. Cadernos FGV DIREITO RIO. Clínicas; 5 [Rio de Janeiro’s FGV Law Series – Clinical]. Rio de Janeiro, Brazil: Escola de Direito do Rio de Janeiro da Fundação Getúlio Vargas, 2016;58.

21. Turchetto M, Vanin S. Forensic entomology and globalisation. Parassitologia 2004;46(1–2):187–90.

22. de Lourdes C-BM, Hernández-Cort
es R, D
ıaz-Torres P, Niderhauser-García A, Ancer-Rodríguez J, Jaramillo-Rangel G, et al. Identification of human remains by DNA analysis of the gastrointestinal contents of fly larvae. J Forensic Sci 2013;58(1):248–50.

23. Campobasso CP, Linville JG, Wells JD, Introna F. Forensic genetic analysis of insect gut contents. Am J Forensic Med Pathol 2005;26(2):161–5.

24. Byard RW, James RA, Heath KJ. Recovery of human remains after shark attack. Am J Forensic Med Pathol 2006;27(3):256–9.

25. Adriaens D, Aerts P, Verraes W. Ontogenetic shift in mouth opening mechanisms in a catfish (Clariidae, Siluriformes): a response to increasing functional demands. J Morphol 2001;247(3):197–216.

26. Wassenbergh SV, Aerts P, Adriaens D, Herrel A. A dynamic model of mouth closing movements in clariid catfishes: the role of enlarged jaw adductors. J Theor Biol 2005;234(1):49–65.

27. Michaud CL, Foran DR. Simplified field preservation of tissues for subsequent DNA analyses. J Forensic Sci 2011;56(4):846–52.