Forensic Entomology PDF

FORENSIC ENTOMOLOGY EDITOR Hakan Bozdogan FORENSIC ENTOMOLOGY EDITED BY Hakan Bozdogan Luke Chinaru Nwosu AUTHORS Ebenezer Okechukwu Emeribe Hakan Bozdogan Luke Chinaru Nwosu Kayode David Ileke Maduamaka Cyriacus Abajue Olutayo Modupeola Adedokun Copyright © 2023 by iksad publishing house All rights reserved. No part of this publication may be reproduced, distributed or transmitted in any form or by any means, including photocopying, recording or other electronic or mechanical methods, without the prior written permission of the publisher, except in the case of brief quotations embodied in critical reviews and certain other noncommercial uses permitted by copyright law. Institution of Economic Development and Social Researches Publications® (The Licence Number of Publicator: 2014/31220) TÜRKİYE TR: +90 342 606 06 75 USA: +1 631 685 0 853 E mail: [email protected] www.iksadyayinevi.com It is responsibility of the author to abide by the publishing ethics rules. Iksad Publications – 2023© ISBN: 978-625-367-368-0 Cover Design: İbrahim KAYA October / 2023 Ankara / Türkiye Size = 16x24 cm CONTENTS PREFACE Hakan Bozdogan Luke Chinaru Nwosu……………..…..……………...………..…………….……..1 SECTION I FORENSIC ENTOMOLOGY CHAPTER 1 FORENSIC ENTOMOLOGY: MEANING, HISTORY, SCIENCE, AND APPLICATIONS Maduamaka Cyriacus Abajue....................................................................….3 CHAPTER 2 FORENSIC INSECTS: CHARACTERISTICS AND ECOLOGY Maduamaka Cyriacus Abajue Luke Chinaru Nwosu Hakan Bozdogan............................................................................................19 CHAPTER 3 SPECIALISED TOPICS IN FORENSIC ENTOMOLOGY Maduamaka Cyriacus Abajue Luke Chinaru Nwosu ………………………………………………………..……53 SECTION II ASPECTS OF STORAGE ENTOMOLOGY CHAPTER 1 STORED PRODUCTS ARTHROPOD PESTS Kayode David Ileke Luke Chinaru Nwosu ……………......................................................…..……67 CHAPTER 2 CONTROL OF STORED PRODUCT ARTHROPOD PESTS Kayode David Ileke Luke Chinaru Nwosu Ebenezer Okechukwu Emeribe..…….....................................................….…75 SECTION II CHAPTER 1 CROP PRODUCTION AND ASSOCIATIONS WITH INSECT PESTS Ebenezer Okechukwu Emeribe Olutayo Modupeola Adedokun ……………………………………….…………93 CHAPTER 2 PRINCIPLES AND PRACTICES OF PEST CONTROL Ebenezer Okechukwu Emeribe Kayode David Ileke Maduamaka Cyriacus Abajue ….........................................................….…109 1 | FORENSIC ENTOMOLOGY Preface Forensic Entomology is an emerging discipline that has garnered considerable attention in recent years. It holds great promise in providing evidence that can illuminate many unsolved murders or plots. This rare challenge of our century is a new branch of science. The book, comprising three sections, offers comprehensive information on the history of forensic entomology, its applications, and standard protocols. Additionally, the book includes a list of insect species that can be utilized in forensic entomology cases, categorized by order and family. This comprehensive guide is expected to broaden the knowledge of both undergraduate and graduate students. Advancing Through Applied Science and Technology| 2 3 | FORENSIC ENTOMOLOGY SECTION 1 CHAPTER 1 FORENSIC ENTOMOLOGY: MEANING, HISTORY, SCIENCE, AND APPLICATIONS Maduamaka Cyriacus abajue1 1 Department of Animal and Environmental Biology, University of Port Harcourt, Nigeria. FORENSIC ENTOMOLOGY| 4 5 | FORENSIC ENTOMOLOGY Meaning of Forensic Entomology Forensic entomology is a science that utilizes insects as evidence in courts. This science involves not only entomologists but also those who provide information about the proper uses of chemicals or pesticides, those who control fly populations in animal waste, and those who can ascertain insect damage to structures and the contamination of food by insects. Cases typically referred to in civil courts that require evidence from forensic entomology are quite common. Hence, this study seeks a precise understanding of forensic entomology which is concerned with the use of insects during the investigation of questionable deaths or other legal cases. Forensic entomologists are the main experts who are expected to present an investigative report or testimony in a law court by using their knowledge of entomology. Basics of Forensic Entomology Forensic entomology is simply defined as the study of insects in forensic science. It is the use of the knowledge of insect biology to legally investigate cases of questionable deaths of humans and animals. In some cases, other arthropods are incorporated into the examination to achieve a similar goal. Terrestrial and aquatic insects have been studied with a view to understanding forensic entomology. Hence, forensic entomology is not restricted to land because it also examines insects in water (Anderson, 1995; Keiper and Casamatta, 2001; Hobischak and Anderson, 2002). In practice, terrestrial insects have received wider attention than aquatic insects. Similarly, corpses recovered in outdoor scenes have received broader forensic attention than indoor scenes. However, the examination of the corpses found indoors is not limited to insect infestations yet they constitute the biggest part of it. Recently, forensic entomology has evolved into entomotoxicology, which is a novel approach in forensic toxicology that alternatively carries out the analysis of toxins on insects that have fed on decomposing cadavers. For a better interpretation of crime scenes, the forensic entomological investigation of mysterious deaths critically evaluates the micro- environmental variables at crime scenes to elucidate how the variations affect the presence, development, and diversity of insects to estimate when the death occurred. Insects are known from ancient times to colonize both invertebrates and vertebrate carrions. Casual observations of these allogenic communities FORENSIC ENTOMOLOGY| 6 reveal a diverse group of insect species. Thus, when the taxonomy of insects became a core discipline in biosciences, the identity of every organism based on morphological attributes helped specific insects associated with vertebrate carriers to be identified. However, in relation to forensic science, insects, especially fly larvae, were largely ignored during autopsy as they were regarded as disgusting elements of decay (Catts and Goff, 1992). It is noteworthy that immediately after death, insects will be attracted to the body often within minutes or hours depending on the location of the dead body and the season of the year unless their access to it is intentionally blocked. Interestingly, insects constitute numerically the largest invertebrate fauna on earth, and hence they are more diversified than other invertebrates. This diversity makes insects available in every ecosystem. The groups of insects that are useful in forensic investigations include the true flies in the following families: Calliphoridae, Muscidae, Sarcophagidae, Beetles, Cleridae, Dermestidae, Histeridae, Staphylinidae, etc. The immature stages of flies are one of the true carrion feeders. In some beetles, both the immature ones and the adults will eat the body. The groups that consume the body are referred to necrophagous insects (Smith, 1986). Other families of insects found on corpses or cadavers are predators or parasites of necrophagous insects. Historical Perspectives of Forensic Entomology The book “ The Washing Away of the Wrongs: Forensic Medicine in the Thirteenth-century China” written by Sung T’zu in 1247 and translated into English by B.E. Mc Knight in 1981 is one of the books of reference on the origin and application of knowledge of insects in a murder case. It was reported in the book that a man was murdered in a rice field after being inflicted with multiple injuries. It was suspected that the injuries were caused by a sharp metal object. The investigator of the murder asked the suspects (rice farmers) to assemble their sickles on the ground. One of the sickles attracted blowflies, probably because of the traces of blood tissues on the sickle. The owner of the sickle was interrogated and he confessed to the killing of the man. Research findings have authoritatively established or at least clarified the understanding of insect metamorphosis, especially that of flies, which disproves the theory of spontaneous generation. The knowledge of the insect life cycle is the core of forensic entomology which remains effective in law court. To exemplify, Bergeret (1855 as cited in Amendt et al., 2004) reported that the French courtroom witnessed the first application of forensic entomology in 1850. In that report, it was written that the mummified 7 | FORENSIC ENTOMOLOGY child was found behind a chimney during the house renovation. The insects found on that mummified corpse were admitted as evidence that the current occupants were not the culprits and they were acquitted. During that time under review, forensic examiners did not have clear knowledge of insect biology but rather their perceptions were based on casual observation. Yovanovich and Megin were recognized as the first forensic examiners because they evaluated insect succession on corpses, properly establishing the science of forensic entomology (Amendt et al., 2004). Dead animals, including human corpses, begin to deteriorate minutes after death due to the changes in the physiological circulation which have come to a halt, thus leading to putrefaction. Insects especially flies are accepted as the evidence of this deterioration since they are the first to detect the change and arrive on the carrions. In addition, beetles arrive on the carrions a few days after death to feed on the soft and dried tissues which aid to skeletonize the carrions (Abajue et al., 2013). Smith (1986) categorized these insects into four carrion ecological communities: 1. Necrophagous insects- these are insects that feed on the carrions. 2. Predators and parasites of necrophagous insects- these are insects that solely feed on other insects or arthropods on the carrion. 3. Omnivorous insects- these include insects such as wasps, ants, and some beetles feeding on both the carriers and their colonizers. 4. Adventive species- these are insects such as springtails and spiders that use the corpse as an extension of their environment. Forensic entomology utilizes insects in the first two groups. They are mainly insect species from Coleoptera and Diptera Orders. Their succession on carrions/corpses can be distinguished into different phases over the various stages of decomposition despite being controversial. About taxonomic composition and facts, African carrion communities comprised at least three hundred species of arthropods in about forty taxonomic families, primarily of insects. These figures can be compared with the lists from the Holarctics (Payne 1965; Smith, 1986) and the Neotropics (Carvalho and Mello-Patiu, 2008; Almeida and Mise, 2009), which were reviewed by Villet (2011). There are some challenges in the study of insects associated with carrions in Africa, especially in Nigeria. One of them is the proper identification of insects at the species level based on the morphological features of the adults. While very few of the identification keys can be FORENSIC ENTOMOLOGY| 8 meticulously found, the keys to the larval stages may not be found though meticulous work. The challenge is a heavy liability that poses an enormous task on forensic entomology as it does right now and makes few carrion researchers rear the larvae to the adult stage. However, this procedure may be hampered as the larvae may die during the rearing process. On the contrary, developed countries in the United States and the United Kingdom have developed molecular methods of identification, enabling the identification of all stages including eggs, thereby linking (with ease) the immature stages to adult identity. This calls for molecular advancement in places where crude methods of identification are still very much operational. The Science of Forensic Entomology and Its Applications The goal of forensic entomology is primarily to determine elapsed time since death. Thus, in the last twenty years in developed countries, forensic entomology has become more and more common in police investigations. Most cases that involve a forensic entomologist are the ones that have stayed up to 72 hours and beyond because insect evidence is often the most accurate and sometimes the only method to determine the elapsed time since death after the first three days. There are two main approaches to using insects to determine elapsed time since death: ▪ Using maggot age and development. ▪ Using successional waves of insects. Any approach used is determined by the conditions surrounding each case. In general, the first approach is used when the death occurred less than a week before discovery. The second approach is used when a corpse is between a week to a month, a year, or more. The first approach is considered by using maggot age and development which can give a date of death accurate to a day or less, or a range of days, and is used in the first few weeks after death. Maggots are immature stages of dipteran flies. The flies used in this method are those that arrive first on the corpse/cadaver. They are mainly the Calliphorids (blowflies) that arrive on a corpse immediately after death. They lay their eggs on the orifices of the corpse/cadaver or in a wound if present. The development of the eggs into adults follows a predictable cycle. The eggs laid on a corpse/cadaver by flies hatch into the first larval instars after a specified period. The larvae will begin to feed on the corpse/cadaver until 9 | FORENSIC ENTOMOLOGY they molt into second larval instars. They will continue to feed on it and molt into third larval instars. The third larval instars will feed on the corpse/cadaver for a while and stop feeding. They will begin to wander away from the corpse/cadaver into the clothes or the soil or nearby debris for pupation. The non-feeding wandering maggots are called the pre-pupae. At this stage, they lose their outer skin but remain inside the skin. The outer skin hardens into a hard protective outer shell, or pupal case, which safeguards the insect as it transforms into an adult. The freshly formed pupa is usually pale in color but darkens to a deep brown after a few hours. After a few days, an adult fly will emerge from the pupa and the cycle will begin again. When adults emerge, it leaves behind empty pupal cases as evidence of successful development and emergence. Each of these developmental stages (egg, larva, and pupa) takes a set of recognized times. This identified time frame is based on the availability of food resources and the temperature. However, food availability is not usually a limiting factor in the case of a human corpse. Note that insects are “cold-blooded” animals, and therefore their development is extremely temperature dependent. Hence, their metabolic rates are increased with the increased temperature resulting in a faster rate of development. Consequently, the duration of development is reduced linearly with the increased temperature and vice versa. When analyzing the carrion community, the oldest stage of insect (larva) on the corpse and the temperature of the region in which the body was discovered will lead to a day or a range of days on which the first fly laid its eggs on the corpse. This will in turn lead to a day, or a range of days during which the death occurred. For instance, if the oldest insect (larva) is estimated to be 5 days old, then the decedent is estimated to have been dead for at least 5 days. This method can be used until the first adults from the first batch of eggs begin to emerge. Beyond this period, it is not possible to determine which generation is present. Therefore, after the first batch of blowfly generation has been completed, the time of death is determined using the second approach that is centered on the use of insect succession. The second approach is centered on the fact that a human body, or any kind of carrion, supports a very fast-changing ecosystem going from fresh decay stage to dry decay of bones in a matter of weeks or months depending on the geographical region. During the decomposition stages, the remains go through rapid physical, biological, and chemical changes. The different stages of FORENSIC ENTOMOLOGY| 10 decomposition are attractive to different insect species. Some species of flies are often the first witnesses to a crime. They usually arrive within less than the first 24 hours of death if the season is suitable and the cadaver is not intentionally covered to prevent flies from accessing the body. The first groups of flies are the calliphorids (blowflies) or the muscids (houseflies), especially in a closed-door scene. Other insect species are not interested in the corpse when the corpse is fresh due to their nutritional and reproductive requirements but are only attracted to the corpse later. Probably due to protein fermentation of the body’s tissues other insect faunas especially the beetles, begin to arrive successively. Some of the insects are not directly attracted to the body but arrive as predators to feed on the other insects on the carrion body. Many insect species observed at each stage of decomposition as well as another group of insects overlap with the ones adjacent to them somewhat. Therefore, with the knowledge of the regional entomofauna and times of cadaveric colonization, the insect assemblage associated with cadavers can be analyzed to determine a window of the time when the death occurred. This method is used when the decedent has been dead for a few days to a year or several years in some cases, with the estimated window of time that extends as the time passing since death increases. It can also be used to indicate the season of death, a dry season, a rainy season, etc. Thus, knowledge of insect succession is required for this method to be successful. 1. ESTIMATES OF TIME OF DEATH In medicine, a medical pathologist can reasonably estimate the time of death of a cadaver when the body is not beyond three days. The pathologist’s approach is based on the historical use of features by studying the body condition such as the fall in body temperature and biological parameters such as lividity, rigor mortis, and post-mortem cooling. Others include changes in the chemical constituents of the body, autolysis, and putrefaction. However, beyond approximately three days, all the known pathological information will remain invalid to estimate accurately the time of death of the individual. This time estimate is referred to as the post-mortem interval (PMI). Estimation of time of death (PMI) is very crucial in forensic investigations; hence, an alternative for this challenge is sought. Thus, forensic entomologists present a means of the possible post-mortem interval based upon the life cycle stages of specific fly species recovered from the corpse/cadaver, or the succession of insect fauna present on the body. This entomological estimate can be valid over weeks or years. The indicator of this 11 | FORENSIC ENTOMOLOGY estimate is based on the knowledge that the beginning of the post-mortem interval is considered to coincide with the time when a fly first laid its eggs on the body and its end to be the discovery of the body based on the recognition of life stage of the oldest colonizing species infesting the cadaver. The time taken to reach this stage concerning the particular stage of decomposition gives an accurate measure of the estimated length of time the person or the animal has remained dead. This may be the best estimate that is alternatively available. Stages of Decomposition of a Corpse/Cadaver The stages of decomposition of a corpse/cadaver vary based on the zoogeographical differences of the ecosystem and other exogenous parameters. However, there are three identified processes in corpse/cadaver decomposition. These are autolysis, putrefaction, and digenesis. In autolysis, enzymes such as lipases, proteases, and carbohydrates begin to digest the cells of the body. This causes the cells to naturally break down. This process is rapid within the organs such as the brain and liver (Vass, 2001). The cells that have broken down become nutrients that serve as a food source for bacteria. In putrefaction, tissues are broken down by bacteria. This process leads to the emission of gases such as hydrogen sulfide, sulphur dioxide, carbon dioxide, methane, ammonia, and hydrogen. In addition, anaerobic fermentation occurs when the volatile fatty acids, propionic and butyric acid, are formed. When the cadaver body undergoes decaying, protein sources are broken down into fatty acids by bacteria. Fatty acids and other compounds such as skatole, putrescine, and cadaverine are significant members of these decomposition products (Vass, 2001). However, these volatile compounds may be absent on buried bodies (Vass et al., 2004). In digenesis, skeletal bones decompose after the soft tissues are removed. Skeletal materials composed of organic and inorganic remains are further broken down by environmental conditions and are finally reduced to components of the soil. However, skeletal decomposition is temperature- dependent. Note that the three decomposition process is synonymous with every corpse/cadaver irrespective of its location on land or in water. On land, a corpse/cadaver can be allocated to one of the five identified post-mortem conditions which can be linked to different waves of FORENSIC ENTOMOLOGY| 12 insects or arthropod colonization. However, no obvious distinction is observed from one stage of decay to the next. Moreover, no stage of decay has a fixed duration; each stage can be associated with a specific assemblage of insects. The sequence of this insect colonization on a corpse/cadaver remains universally constant at the family level among locales. However, at the species level, the colonization of a body is influenced by environmental and location-specific factors depending on the zoogeography of the region (Early and Goff, 1986). Hence, post-mortem changes are broadly recognized in these stages: ▪ Fresh stage: This stage commences from the moment of death to the first sign of bloating of the body. The first specimens to arrive on the corpse/cadaver are the calliphorids (blowflies). However, the species may vary concerning zoogeographical locations and seasons. ▪ Bloated stage: This second stage is the continual breaking down of the body due to putrefaction and perhaps it is the easiest stage to recognize. Anaerobic bacteria cause the nutrients to metabolize and, as a result, generate gases causing the corpse/cadaver to bloat. The initial evidence is the abdominal swelling that precedes the whole body’s becoming stretched like an air-balloon. This stage of decay attracts more blowflies probably as a response to the odor of the breakdown gases. Beetles such as Histerids and Staphylinides may be attracted to the body at this stage because of fly eggs and maggots that serve as ready meals for beetles. As they are predators, they can alter the interpretation of the range and life stages of the existing insects on the corpse as they predate on the maggots or remove pupae (Smith, 1986). ▪ Active/Wet decay stage: This third stage is recognized as an inflated and ruptured body because the skin of the body breaks up and starts to slough from the body. The sloughing allows the decomposition gases to escape, and thus the distended body begins to subside gradually while putrefaction goes on. At the peak of putrefaction, there occurs fermentation, and butyric and caseic acids are produced (Gennard, 2007). After that, a period of advanced putrefaction appears involving ammoniacal fermentation of the body to which different species of insects are attracted. These insects include clerids, dermestids, 13 | FORENSIC ENTOMOLOGY histerids, staphylinids, and maggots of calliphorids, ulidiids, and stratiomyids (Abajue, 2016). ▪ Post/Dry decay stage: This fourth stage may be the remnants of the previous stages which include the skin, cartilage, and bones as well as the gut contents. This stage of decomposition may be omitted during the rainy season in eastern Nigeria (Abajue, 2016). The evidence of this stage is the conspicuous number of beetles and their juvenile stages. Blowfly maggots are as well drastically reduced leaving behind their pupae while ulidiid and stratiomyid maggots become preponderance beneath the cadaver body (Plates 1, 2, 3, 4). Plate 1. A fresh pig cadaver exposed to insect infestation. FORENSIC ENTOMOLOGY| 14 Plate 2. A Bloated pig cadaver infested with fly eggs and larvae. Plate 3. Active decomposing pig cadaver infested with fly larvae and beetles. 15 | FORENSIC ENTOMOLOGY Plate 4. Dry decomposing pig cadaver infested with beetles and their larvae. Plate 5. A skeletonizing pig cadaver infested with beetles and their larvae. FORENSIC ENTOMOLOGY| 16 Skeletonization This stage is recognized by the absence of body tissues except for the hairs and the bones. Insects found at this stage are mostly keratophagous beetles (clerids and dermestids). Decomposition is finalized at this stage as further breakdown is best described in terms of the decay of individual components of the body such as the bones of the feet, skull, vertebral column, and ribs. 2. EVIDENCE OF PHYSICAL ABUSE One major contribution of forensic entomology is to provide the estimated time of a questionable death. However, forensic entomology has a role to play in the investigation of other crimes in which the victim is alive because insects can also provide evidence in cases of neglect or abuse. Blowflies, for instance, are attracted to odors emanating from ammonia due to urine or fecal contamination. They tend to infest a baby who is deprived of regular diaper/nappy changes or an incontinent old individual who has not been assisted to maintain sufficient hygiene (Gennard, 2007). Flies, if allowed access, may lay eggs in the clothing or on the skin. If the eggs are not discovered, they will hatch into maggots that start to feed on the flesh or in wounds, ulcers, or any of the orifices of the body. This is referred to as myiasis. With time the flesh will be eaten away and the region may be further infected by bacteria as well as invaded by other insects. Similarly, insect attacks can also happen to animals such as rabbits, pigs, dogs, and sheep. These animals can be victims of a fly strike because of urine or fecal material attached to the furs, fleece, or hind quarters through neglect or poor caging and living conditions as well as ill-health reflected by scouring. Such cases are considered to be instances of physical abuse since victims are unable to remove the eggs or maggots themselves. The effects can be serious and require attention from a veterinarian, otherwise, they may lead to the death of the animal or require its euthanasia. Therefore, caution has to be taken in making assumptions about the existence of physical abuse or assault before death. 3. ENTOMOTOXICOLOGY Entomotoxicology is a new area of entomology whereby insect evidence on a corpse is analyzed to determine whether drugs or toxins were used before death. The insects that feed on a corpse/cadaver are a good 17 | FORENSIC ENTOMOLOGY potential reservoir of undigested flesh from the corpse as the tissue from the corpse can retain drugs for some time in parts or in a whole that had been consumed by the deceased before death and which may even be the cause of death. Thus, drugs may be recovered if present by analyzing the insects that have fed on the corpse. This branch of forensic investigation is very necessary when the decomposing body is difficult to toxicologically examine due to a lack of appropriate sources such as tissues, blood, or urine. At this stage, immature insects such as maggots and pupae of flies may serve as an alternative material (Nolte et al., 1992; Goff and Lord, 1994; 2001; Introna et al., 2001 and Campobasso et al., 2001). No information up till now has, however, stated clearly the role of drugs or poisons in insect immaturity (Gennard, 2007). In addition, how immature insects bio-accumulate or eliminate drugs or poisons is not yet understood (Gautam et al., 2013). However, numerous empirical and case studies have reported that certain drugs of abuse have the potential to increase the rate of development of insects feeding on a poisoned corpse or cadaver (Goff et al., 1989; 1991; O’Brien and Turner, 2004). Similarly, some poisons in the form of pesticides may decrease the rate of development of cadaveric insects (Bourel et al., 1999; Gunatilake and Goff, 1989). Therefore, overestimation or underestimation is possible if such variability is not considered when determining the PMI of a corpse. Therefore, it is necessary to evaluate the life style of the victim and pay attention to the antemortem mood swings of the victim. 4. INSECTS AND DRUG TRAFFICKING Forensic entomology in a broad sense is not restricted only to the questionable deaths of humans but includes inquiry into poaching activities in game reserves as well as tracing the origin of plant materials legally prohibited. It has been reported that two separate cannabis seizures along with eight Asian species of beetles, as well as wasps and ants in New Zealand, were clear evidence of the role of insects in drug trafficking. The beetles were identified as belonging to the families of Bruchidae, Carabidae, and Tenebrionidae. Considering the geographical distribution of the insects and the level of overlap of their distributions, entomologists concluded that the cannabis came from the Tenasserim region, between the Andaman Sea and Thailand. In affirmation, one of the two suspects confessed on the basis of this evidence (Crosby et al. 1986). This kind of forensic FORENSIC ENTOMOLOGY| 18 information is considered to be useful to customs, excise officers, and drug law enforcement agencies. 5. INSECTS IN FOOD INDUSTRY Stored product insects may be eaten along with food or seen on commercial foods. In many instances, this is unacceptable to the consumer and can be taken as food contamination or poisoning. Stored product insects such as saw-toothed grain beetle may be found in cereal packages. Others include wireworms which may be sold along with freshly cut lettuce or it may be processed into lettuce and tomato sandwiches. In developing countries where fish and meat processing is done locally, insects are attracted to infest them because they are left in the open place to dry. Hence, fish and meat bought from the open market are not always free from beetle and fly larvae. Undoubtedly, these insects may potentially cause illness when they are eaten alongside fish or meat. Hence, forensic entomologists do entertain questions to submit their professional views in civil cases relating to the food industry where synanthropic insects are found to contaminate food. However, this does not make insect diet (entomophagy) an unhealthy practice because insect species are consumed globally. For instance, Lethocerus indicus Le Peletier & Serville, a giant water bug is a delicacy in South-eastern Asia while chocolate-covered bees and canned fried grasshoppers are sold in the UK and North America. Presently, Thailand is known for the mass production of crickets sold in tins for food (De Foliart, 1988). In Africa, wide varieties of insects at different stages of development are eaten extensively to augment the protein requirements of children and lactating mothers. 19 | FORENSIC ENTOMOLOGY CHAPTER 2 FORENSIC INSECTS: CHARACTERISTICS AND ECOLOGY Maduamaka Cyriacus Abajue1 Luke Chinaru Nwosu2 Hakan Bozdogan3 1 Department of Animal and Environmental Biology1 & Department of Crop and Soil Science 2 University of Port Harcourt, Nigeria 3 Department of Plant and Animal Production, Kırşehir Ahi Evran University, 40100, Kırşehir, Turkey. FORENSIC ENTOMOLOGY| 20 21 | FORENSIC ENTOMOLOGY Overview Identifying the identity of insects that are of forensic importance is one of the first crucial steps in ascertaining the role of the species infesting the corpse/cadaver in addition to their habitats and environmental requirements. This endeavor requires knowledge of insect structure as well as an understanding of various parts of insects in general. Scientific keys to these identification requirements have been written by insect taxonomists. Paying attention to the keys will help entomologists easily identify the insects morphologically. Moreover, newer and better methods of identification now exist in the form of DNA analysis which even takes care of the immature stages as an advantage over the former. However, the two methods are still within the scope of entomology and are acceptable and valid in a law court. The knowledge of college biology still refreshes our memory that insects are arthropods with six legs (three pairs of legs). The whole body structure of insects is divided into three sections; the head, the thorax, and the abdomen. Each of the sections has three dimensions. The top part view is referred to as the dorsum; the underneath is the sternum while the sides are referred to as pleura (pleuron-singular). The head section is a capsule that contains the mouthparts and the sensory organs such as the eyes and antennae (the feelers). The thorax section is segmented into three parts. The first segment proximal to the head is called the prothorax which bears one pair of legs. Each of the legs has five sections starting at the point nearest to the body called the coxa which is followed by the trochanter, the femur, the tibia, and the tarsus in that order. The tarsus ends up with tough tiny and spiny-like structures called claws. The second segment of the thoracic body is referred to as the mesothorax which bears another pair of legs. It also bears a membranous pair of wings at least in a particular stage of its life history. The last segment of the thoracic body is referred to as the metathorax which also bears the last pair of the insect legs in addition to the second pair of wings (which is replaced with organs called halters in dipteran flies). These characteristic features such as the antennae in various forms and shapes, the legs, and the membranous wings aligned with serially arranged veins are taxonomic features in insect identification. Precise and accurate identification is such a vital step in forensic entomology that the help of expert taxonomists should be consulted in cases of doubt about the identity of a particular insect species. Hence, it is recommended that forensic researchers intending to use entomological evidence as a career or profession FORENSIC ENTOMOLOGY| 22 in a forensic investigation should familiarize themselves with cadaveric insects, especially the Calliphorids (blowflies). Moreover, special attention should be given to the identification of insect larval stages of blowflies because their diagnostic features are not readily covered by literature and should be reared by adults. This is achieved by engaging in good observation and careful documentation which will help to produce the collection of entomological data as a standard routine in forensic inquiry. The more cautious the investigation is, the more accurate and precise the method will become. Insects (hexapods) by systemic identification are in the Phylum Arthropoda. The class is divided into several groups called orders. Each order is further divided into several families. Each family also contains some genera and each genus may have more than one species. Diptera The order Diptera contains true flies such as blowflies, houseflies, fleshflies, horseflies, and craneflies. Others include midges and mosquitoes. Adult flies generally have just one pair of wings. The second pair is modified as organs for balancing called halteres. Flies such as blowflies, houseflies, and fleshflies are synanthropic and, certainly, the most important insects associated with corpses/cadavers or carrions. The larval stages are generally called maggots. Fly larvae have distinct heads but with no defined thoracic legs except the abdominal prolegs. It is advisable to consult the bibliography in the Diptera catalogue for each Zoogeographical region. This, however, limits entomological studies in developing countries. 23 | FORENSIC ENTOMOLOGY Phylum Arthropoda Class Insceta/Hexapoda Order Diptera Suborder Suborder Nematocera Brachycera Infra order Infra order Muscamorpha Muscomorpha Schizophora Aschiza Calyptrate Family Calliphoridae Genus Chrysomya Species Chrysomya albiceps (Weid.) Figure 1. Schematic Classification of Chrysomya albiceps Weid. (blowfly) FORENSIC ENTOMOLOGY| 24 Calliphoridae This family constitutes the blowflies that are of greatest importance in forensic entomology for determining the time of death (PMI) of human and animal cadavers. The family consists of over a thousand described species and is well-represented in all zoogeographical regions. A large number of blowfly species develop in decaying organic remains including cadavers. Thus, bionomics of blow flies has discovered different species including species of myiasis importance in the Old World (Zumpt, 1965). Moreover, the taxonomy of blowflies is not easy as many species of forensic importance are continuing to get attention more than the already reported species in the literature. Thus, for accurate data, forensic entomologists should study in detail the cadaveric flies in their zoogeographical region with references contained in the appropriate Diptera catalog. Important genera in this family include the Calliphora, Chrysomya, and Lucilia with different species restricted to different geographical regions and as well influenced by seasons and environmental habitats. One of the most common species in eastern Nigeria is Chrysomya albiceps Weidmann with a characteristic large metallic blue or green fly. It has a yellow or white thoracic spiracle. It has dark bands across its abdomen. Its legs are dark. The larvae of Chrysomya species are hard to distinguish in appearance except for employing mitochondria DNA analysis. Muscidae Muscid flies such as houseflies, face-flies, stable-flies, sweat-flies, and others have worldwide distribution. Several species of muscid flies are of medical importance due to their relationship with man and his dwellings. They feed on excrement, garbage, compost, sewage, carrion, etc., and may become mechanical transmitters of diseases when they settle on foodstuffs intended for human consumption. Because of their synanthropic habit, some muscid flies are likely to be involved in medico-legal cases, particularly in domestic situations. Musca domestica Linnaeus, for instance, has followed humans around the world. The adult is characteristically distinguished from other house flies by possessing a sharply angled outer portion of the fourth long-wing vein. The fly is mouse-grey in color with an average of 6-7 mm in size (Smith, 1986). It is recognized by four narrow black stripes along its thorax. M. domestica L. found in Nigeria is distinguished by a characteristic yellow abdomen. 25 | FORENSIC ENTOMOLOGY Sarcophagidae Sarcophagid flies are a large family of diverse distribution. The members are commonly referred to as flesh flies. The adults are generally large (4-16 mm long). The body is silvery-grey and has black strips at the thorax and a tessellate or spotted abdomen with strong bristles and reddish eyes (Smith, 1986). Flesh flies are found to associate with carrions. They prefer cadavers exposed to the sunlight. Sarcophaga inzi Curran, for instance, was found to visit pig carrion within three minutes of exposure in an open fallow land (Abajue et al., 2013 and 2014). Females are viviparous because they deposit active first- instar larvae. Sarcophaga species in the tropics are very similar in appearance and difficult to identify both as larvae and adults. Larvae should be reared into adults before identification. Many of the species develop in excrement, carrion, or other decomposing remains. S. inzi Curran may come before the forensic entomologist (personal observation). Rearing the larvae associated with carrions through adults (Abajue, et al., 2017) is very essential because the diagnostic feature described by Smith (1986) for Sarcophaga haemorrhoidalis (Fallen) is morphologically similar to that of S. inzi (Curran). Stratiomyidae Stratiomyids are fairly large flies measuring about 5-15 mm in length. They are referred to as soldier flies because of the ‘armament’ of spines on the scutellum of some species. Their colors range from brilliant metallic green and purple to black and yellow. Their bodies are generally flat with discal cells in their wings. The larval stages are found in rotten woods, under bark and in dung, lavatories, and compost heaps and will probably feed on any decaying remains of animal or vegetable origin. Stratiomyid larvae are distinguished by their roughened appearance, like shark skin. They have flattened mouthparts moving vertically parallel to each other like a pair of hooks (Smith, 1986). One of the species generally found to associate with carrion decomposition in Nigeria is Hermatia illucens Linnaeus (Ekanem and Dike, 2010; Ekrakene and Iloba, 2011; Abajue et al., 2013 and 2014). Bionomics of the carrion community is continually dominated by the assemblage of flies mainly during the active decompositions as primary necrophages. The flies usually recovered from carrions but not forensically discussed for being used to estimate the time of death include species in the following families: Coleopidae, Drosophilidae, Dryomyzidae, Ephydridae, FORENSIC ENTOMOLOGY| 26 Fanniidae, Heleomyzidae, Milichiidae, Phoridae, Piophilidae, Psychodidae, Sepsidae, Sphaeroceridae, Syrphidae, Trichoceridae, etc. The forensic importance of these and other families not covered in this book is that their recovery of the corpse will give clues about the location of the crime scene as well as the season. Coleoptera Coleoptera Order constitutes the group of insects generally called beetles and weevils. Beetles are distinguished by the possession of hardened fore-wings that forms elytra or wing cases which usually cover the abdomen, thus giving them an armor-plated appearance. Most beetles can fly but they are usually found on the ground, in the soil, and under the bark of trees and leaf litter among organic debris. Many beetles have been reported to be associated with carrions. In literature, however, many of them are predators while few of them are true necrophages. Both the adults and larvae are found on carrions but their feeding habits may differ. Nevertheless, beetle species have received more forensic discussion than fly species only because of the fact that the greater mobility of flies facilitates them to reach a carrion/cadaver before the beetles. In addition, the faster rate of larval growth of flies enables them to complete their development before the carrion resource is exhausted. This competition suggests why carrion beetles have behaviourally evolved to succeed the flies at a later drier stage (Crowson, 1981). While larval predating beetles such as histerids, staphylinids, etc. regularly exist on carrions during the wet decay stages, core keratophagous beetles (clerids and dermestids) also arrive on carrions during wet decay stages (Abajue et al., 2013; 2015), yet they tend to avoid the wet tissues. Cleridae Clerids are small beetles with hairs, especially on the thorax. These beetles are one of the stored product pests of hides and skin especially on dried fish in eastern Nigeria (personal observation). They are mainly bluish- green. Some species are distinguished by having a red color on the thorax and the anterior part of the abdomen. Both the adults and larvae are probably predaceous on maggots. They constitute the major carrion/cadaveric fauna during the drier stages of decay. The most frequently submitted clerid in forensic entomology is the genus Necrobia (Necrobia ruficolis Fab. and Necrobia rufipes Deg.). 27 | FORENSIC ENTOMOLOGY Dermestidae Dermestids are moderately small-sized beetles densely covered with short hairs or scales which are often conspicuously grey. Both the adults and larvae specifically feed on dried fish, hides, skins, furs, etc. The larvae are characteristically distinguished by their brownish urticating hairs. Species of forensic value as frequently reported are the genus Dermestes spp. Histeridae Members of this family usually have moderately short elytra that leave the last two abdominal segments visible. They are most squat in shape. Their colors range from bluish-green to black. These beetles have been predictably found on carrions during the bloating stages of pig carrions in eastern Nigeria. Their presence on carrions synchronizes at least the first instars of blowfly larvae which they frequently predate on. Species in the genus Hister are abundant all through the year in Eastern Nigeria. Staphylinidae Staphylinids are moderately tiny beetles with very short elytra. Many of the species associated with carrion are distinguished by the yellow color of the thorax. They are usually not noticed and observed unless carefully searched for because of their fast disappearance into holes and debris underneath and beside carrions when a slight disturbance is felt on the body. Both adults and larvae are active maggot predators. Two forensic important genera; Philonthus and Staphylinus are usually reported in forensic entomology. Other beetles that have been reported in forensic literature include these families: Carabidae, Geotrupidae, Hydrophilidae, Leiodidae, Nitidulidae, Ptinidae, Rhizophagidae, Scarabaeidae, Silphidae, Tenebrionidae, Trogidae, etc. The assemblage of these beetles has roles to play as indicators of the carrion/cadaver’s environment where they are discovered. The Life Cycles of Insects Associated with Dead Human and Animal The life cycle is the predicted metamorphosis that is exhibited in the development of animals, it gives the general overview of the life forms of an individual, whether oviparous, viviparous, ovoviviparous, or some other forms of development. FORENSIC ENTOMOLOGY| 28 Flies and beetles are among the insects that have life cycles that show four stages of metamorphosis. It means that each stage of the life cycle is unique and different from the other stage. The life cycles of these insects start when the adult females lay eggs which develop into another stage called larvae. Then, the larvae will undergo developmental changes to pupae and adult stages. The estimated duration of each stage and summation of the stages from egg laying to adulthood in both flies and beetles associated with vertebrate decompositions have been documented (Reiter, 1984; Greenberg, 1991; Anderson, 2000; and Grassberger and Reiter 2002). The estimation of these stages of insects on cadavers is the primary hypothetical tool used by forensic entomologists to estimate when the animal died. Flies associated with vertebrate cadavers tend to lay eggs in batches. Egg clumps are laid in the parts of the corpse that offer protection, moisture, and food (Gennard, 2007). It was suggested that Calliphora vicina may lay 2000-3000 eggs in their lifetime (Hinton, 1981). Eggs of blowflies which are the most reported egg samples recovered from vertebrate cadavers are usually very shiny and white with sizes ranging from around 0.9 mm to over 1.50 mm long and 0.3-0.4 mm wide (Rognes, 1991). The emergence of the first instar larvae from the eggs called eclosion is a term also used to describe any form of hatching. However, this may not be observed in the flesh flies (Sarcophagidae) which do not lay eggs on the carrion but deposit first instar larvae. This type of development has been observed in some C. vicina flies in which fertilization takes place without a suitable oviposition site available (Erzinçlioğlu, 1996). The blowfly larvae have been reported to have twelve segments. The blunt posterior end has two brown circular areas on the final segment called the posterior spiracles (Gennard, 2007). The larvae have three larval stages or instars and each stage is distinguishable. The specific life stage of each larva can be identified by the number of slits present in each posterior spiracle. There is one slit in the first instar, while two slits in the second instar and three slits in the third instar are present (Ekanem and Usua, 2000). There is normally a difference in the size of larvae in the three larval stages of blowflies. Nevertheless, size is not an entirely reliable measure of the age of the larvae because it differs according to the amount and quality of the food available on the flesh considered to be an ample repository of food for insects (Gennard, 2007). Larvae in the late third instar tend to stop feeding and become migratory, searching for a drier place for pupation. This is the final 29 | FORENSIC ENTOMOLOGY developmental stage of metamorphosis into the adult stage. This is called the larval post-feeding stage. Usually, the post-feeding larva attempts to bury itself in soil or some other dark locations. They may be found by searching in the first 2-3cm depth of soil at outdoor crime scenes. The pupa is simply the transition stage between larva and adult. It is found inside a barrel-shaped puparium which is the hardened and darkened skin of the final larval instar. The pupal case changes color over time becoming an oval object resembling an uncut cigar in a color between reddish-brown and dark mahogany brown or black (Gennard, 2007). Some attempts have been made to relate the color development of the pupae to post-mortem interval but, to date, the method has not shown great accuracy for the period after the first 24 hours (Greenberg, 1991). At the end of the life cycle, the fly pushes the cap (operculum) out of the pupal case using the ptilinum, a blood-inflated region on the head, and the adult emerges (Gennard, 2007). The immature stages of blowflies and other flies associated with carrion decomposition are poorly documented in comparison to adults. Beetles as well, show complete metamorphosis in their development. Before reaching adulthood from the egg stage, they pass through three to five larval stages depending on species, and a pupal stage. Beetle eggs tend to be oval, spherical, or spheroid in shape usually considered very similar, irrespective of family, and they are mostly found buried in a specially constructed chamber or in the soil while they are pupating (Gennard, 2007). The length of the life cycles of beetles varies depending on the family and species of the beetle. While it takes days in Staphylinids, the Carabids take up to one year to complete one life cycle to adulthood. In some species, the number of instars in the larval stage is not fixed as it is dependent on environmental conditions. Hinton (1945) reported as many as nine instars in dermestids while Smith (1986) noted that there is only one generation of beetles per year (cited in Gennard, 2007). The pupal duration of dermestid species can range between 2 weeks and 2 months and these species tend to overwinter in the pupa chamber due to unfavorable weather conditions or till it becomes late winter (Smith, 1986). The information available about beetles’ life cycles is less detailed than that about flies. Ecology of Insects of Forensic Importance Micro-environmental conditions are ecological variables that define a micro-habitat in terms of vegetation. Soil types as well as the meteorological FORENSIC ENTOMOLOGY| 30 conditions of the area will clearly have a major impact on the types and diversity of insect species present as well as their seasonal availability. Insects that are attracted to vertebrate carrions are influenced by these micro- environmental variables. These variables are of vital importance in interpreting a crime scene and estimating the length of time a body has been dead. Insects may be present or absent on carrion depending on the micro- environmental conditions of the area. Seasonality affects the daily activity patterns of flies which also differ from one geographical location to another. Greenberg (1990) reported that calliphorid flies do not fly in the rain. Hence, seasonal factors such as cold and rainy weather may inhibit or even prevent fly activity and thus delay oviposition (Erzinçlioğlu, 1996). Female insects choose to lay eggs in places on a body that provide sufficient food for the new generation along with protection, moisture, and a consistent microclimate needed for larval development. As a carcass, a human corpse, for instance, decomposes, it offers a food source large enough to support colonies of several different fly species (Gennard, 2007). Archer and Elgar (2003) reported that preferred colonization sites on a carcass changed from the orifices to skin folds after the carcass was exposed to outdoor conditions for 24 hours. These sites include locations such as those between the legs or under the ear pinnae. They concluded that the migration of fly larvae, for instance, to a more favored site was in response to food depletion. Blowflies usually show peaks of oviposition activity in the early afternoon (Baurmgartner and Greenberg, 1987; Greenberg, 1990). This shows that light intensity and over-lapping temperature as well as relative humidity influence oviposition in flies. In laboratory experiments, the developmental times of calliphorids were increased under a cyclical temperature regime compared to the developmental times at a constant temperature (Byrd and Allen, 2001). Variation in the duration of the life cycle occurred at higher temperatures (35 - 45C) where adults failed to emerge and when cultures were kept at a constant temperature of 40C or 10C. This observation indicates that the development of Phormia regina tends to get inhibited when the monthly temperatures reach an average below 10C. Chrysomya albiceps is commonly the initial colonizer of a corpse in Afro-tropical regions and oriental regions from India to China, Central South America, and Southern Europe (Hall and Smith, 1993). It was also recorded 31 | FORENSIC ENTOMOLOGY as one of the two most encountered species in forensic cases in South Africa (Mostovski and Mansell, 2004) where it is recognized as a spring and summer species. This species was considered to fulfill the initial colonizing role played by Calliphora and Lucilia spp. in the temperate zones (Smith, 1986). Chrysomya species have been recorded in Northern France, Austria, and central Europe (Erzinçlioğlu, 2000; Grassberger et al., 2003). The level of interaction of these species with other species rather than the influence of higher temperatures alone is suggested by Grassberger et al. (2003) as an important factor in determining the changes in its distribution. The carrion community presents many instant changes over three- time scales: circadian duration, annual duration, and the duration of decomposition. Being ectothermic, most carrion animals are less active in colder conditions such as at night and in winter. The range of mechanisms affecting community dynamics over the life span of a carrion source is more complex (Villet, 2011). The occurrence of circadian cycles in adult blowflies and flesh flies is important in forensic contexts because it determines the hours when eggs can be laid, and this enables the estimation of post-mortem intervals (PMI). If an animal dies early in the evening, flies will probably not lay eggs on it until the next day, and an estimation of the PMI based on the development of the immature flies will need to take this disparity into account (Villet, 2011). Nocturnal oviposition has never been reported from outdoor sites in temperate climates, but there are several reports from tropical Asia where nocturnal temperatures are higher (Singh and Bharti, 2001, 2008; Villet, 2011). Circadian rhythms also affect the timing of other activities of invertebrates such as the migration of mature maggots from carrion and the eclosion of adults. Thus, Villet et al. (2010) suggested that the circadian impact on the accuracy of such PMI estimates by norming the timing of these developmental landmarks to particular times of the day produces a variable bias and precision that limits the accuracy of such estimates. Similarly, seasonal cycles determine whether a species breeds on carrion at all. The core membership of the carrion community is therefore more strongly affected by a seasonal variation than by the time of the day (Villet, 2011). As earlier stated, temperature truly regulates the seasonal occurrence of many carrion arthropods, producing changes in which species represent their association with one another. In South Africa, Dermestes peruvianus is more common in winter and D. maculatus in summer. Calliphora croceipalpis is characteristically active in winter, Chrysomya chloropyga in spring, and C. FORENSIC ENTOMOLOGY| 32 putoria in later summer. In the case of the flies, these differences in seasonal occurrence are reflected in their thermo physiological tolerances and their geographical distributions (Richards et al., 2009a, 2009b) (Villet, 2011). In a nutshell, the decomposition of vertebrate carrion is generally influenced by environmental conditions. This has however affected the variations in the arrival of some insect families in different geographical regions. For instance, dermestids known to inhabit dry carcasses have been reported sooner on carrions. Van Laerhoven and Anderson (1996; 1999) in North America recorded dermestids 21 days after death when the body was reported to be in early advanced decay. Oliva (2011) in Argentina found them as early colonizers between 10 and 30 days after death. However, reports from Nigeria have reported them on day 4 (Abajue et al., 2013) and day 10 respectively (Ekanem and Dike, 2010; Ekrakene and Iloba, 2011). Ideally, this suggests that data about insect succession on carrion for a particular region where death occurred should be used. Applied Forensic Entomology The best way to respond to a call to visit a crime scene is to get a ready-made carrying container or bag having all the requirements for collecting insect specimens and other entomological valuables. This is to avoid unnecessary delay in responding to a call. If a forensic entomologist is asked to attend a crime scene by the police, he/she needs to collect reasonable entomological samples so that they can be available when they are legally requested by a fellow forensic entomologist to make his/her valuation for the defense. This is very important to be well-equipped to answer questions that must emanate from the reports when presented. Collection of Insects at Crime Scene The collection of insects on a decomposing body at the crime scene is the first and most crucial step in the journey to provide entomological evidence to forensic investigators. Therefore, insects collection equipment that is needed includes plastic or poly-carbonate screw-top sampling jars for both preserved specimens and live cultures, forceps, stepping plates to preserve the scene from contamination, a killing jar containing ethyl acetate, labels, indelible markers with fine points, fine forceps, artists’ paint brushes, an entomological net, killing agents for larvae such as boiling water, and insect preservative. Several preservatives could be used, including 70-80% 33 | FORENSIC ENTOMOLOGY alcohol, KAAD, and Kahle’s solution. Each of these preservatives has its benefits (Gennard, 2007). Bear in mind that live maggots must be recovered from the site; therefore, it is vital to bring some food for them. Pig’s liver or ground beef is the most suitable food. However, research indicates that larvae show varying growth on different body parts. Ground mackerel fish was substituted for pig’s liver and ground beef in Abajue (2012). Any food chosen at a particular time due to proximity and availability should ideally be at room temperature, not frozen or chilled when the maggots are placed upon it. When returning, the cultures should be kept at a low temperature; at least below the base temperature of the maggots is ideal. A mobile refrigerator for the car or van or a cool box with artificial ice blocks would be ideal. A thermometer should be included in the container to ensure that the temperature during transport is known. A carrying box or packaging for the specimens should be included. The sample jars of preserved and live maggots from each site on the body should be retained together in pairs. If samples are collected by a crime scene officer instead of a forensic entomologist, it is better to package and seal the samples to prevent risking their validity. The storage packages can be cardboard boxes. The packages require pin holes and the lids of the culture jars need pin holes too or a porous covering that is firmly attached to the top of the container. Maggots are very artistic in escaping from containers by pushing through a top if it is not tightly secured. When they escape, evidence is lost and thus all efforts become useless. In addition, polythene bags are used to package maggots at a crime scene. Pin holes are made through the bag to avoid a build-up of carbon dioxide, whilst preventing the larvae from escaping. The maggots should be immersed for at least 30 seconds in boiling water to kill them from each site of colonization on the body and fix their maximum length. Boiling water can be brought to a crime scene in a portable thermos flask. Alternatively, prepare on-site hot water using a small camping stove and kettle (don’t forget to go with your matches or gas lighter in this regard). Other prerequisites include studying the general habitat of the crime scene. Observe whether the body is wrapped or covered and how. If the body is indoors, check whether the windows are open or closed. Ascertain the slope of the ground if the crime scene or the body is outdoors. Study the nature of the vegetation if any and give a general description of the scene. Take FORENSIC ENTOMOLOGY| 34 photographs of the scene and record the scene temperature along with the degree of light shed at the scene. Thus, a thermometer should be included in your entomological equipment case. The thermometer should be calibrated so that it reads accurately and does not give readings that must be corrected. Use a digital probe thermometer for safety reasons. However, an alcohol thermometer is preferred to a mercury thermometer. If possible, go with a weather recorder to the crime scene in order to record temperature, light intensity, humidity, wind direction, and speed, which can all be recorded over a specific time. Use a portable thermo hygrometer to record at least the ambient temperature and relative humidity of the scene. Strategy for sampling entomological specimens at crime scenes Eggs Once a crime scene is sealed for investigation and necessary permissions are obtained, the body is thus searched in an orderly sequence. The head is first examined followed by a thorough search of the trunk and later on moving onto the legs and the toes. Any wounds are precisely noted. Once the top side is checked, the body should be turned over and the underneath should be examined. Clothing should be quickly examined on-site, especially the pockets, sleeves, and clothing folds. A more thorough search can as well be extended at the mortuary when the clothes have been stripped from the body. Fly eggs, for instance, are laid in batches and can be mistaken because everything is from yellowish-white mold to sawdust or an encrusting of salt on the body whereas beetle eggs are laid individually and therefore they may be easily missed at the crime scene. Fly eggs are laid in dark, moist orifices of a body (ears, nose, eyes, mouth, anus, or genitalia). Other areas include folds of skin, the back of the ears, the inside of the joint folds, or the parts of the clothing that have absorbed body fluid exudates. Therefore, every part of the body must be thoroughly examined. It is also necessary to attend the post-mortem examination to check further for insects. This is necessary if the body is fully clothed or has been wrapped in some stuff. The clumps of eggs should be collected individually from the body and carefully placed in a container without food. The humidity in the container must be maintained by using a damp paper towel carefully placed on the container to prevent the eggs from getting dry. Every sample should be well tagged with crime scene details such 35 | FORENSIC ENTOMOLOGY as crime scene no., name of the officer in charge, name of the collector, date, item no., and location and description of the scene. These details should be written with indelible ink but not ballpoint ink. The label should be duplicated. One duplicate should be placed on the body of the container while the other one (non-adhesive version) inside the container. This practice of double placement of information inside and outside the container is to limit the chances of losing the information and ending up with a sample of an unknown source. To get the paper label into a container, roll the paper round a pencil and deposit the roll through the neck of the container and it will unroll itself. The label data must be recorded in the scene log book. Larvae Larvae will be found as the body is explored for eggs. They are most likely to be in the orifices and wounds which were inflicted on the body. The larvae should be sampled from each site in batches or sets of 15-30 per jar so that no additional heat or ammonia is produced during delivery. More than one sampling jar per infestation site may be required. The first larval instar is the smallest and most vulnerable of the three larval stages and the larvae, if collected at this stage, can easily die. It is, therefore, necessary to protect them from drying out when collecting and culturing the larvae from a corpse at a crime scene. Boiling or hot water in a portable thermos flask is poured into a container such as a styrene cup or a collecting jar to a depth of 4 cm depending on the volume of the jar and the number of the larvae collected. Larvae which are to be preserved from the specific site are then added to the collecting jar. They are allowed in the hot water for at least 20-30 seconds before the contents of the jar are poured through a small sieve and collected in a large container. Large bottled water container of any choice is ideal to serve such a purpose. The contents of the container when full can be poured into an excavated pit away from the crime scene. Larvae are known to mass together when they reach the late second and third instar stages. These are referred to as maggot masses which are capable of raising the temperature above air temperature and the additional heat can affect the rate of larval development. If a larval mass is observed, it should be photographed and the mass temperature should be recorded in reference to the location being sampled. The temperature of every maggot mass ought to be recorded at each site on the body. This is necessary because FORENSIC ENTOMOLOGY| 36 it can be elucidative in the calculation of the thermal history of the crime scene. Pupae Fly pupa is usually seen at some distance away from the body. These are the third instar post-feeding larvae that have left the carrion resource and can be found in soil about 3-5 cm below the soil surface. They can also be found in pockets, under carpets, in leaf litter, or in any corners and crevices which are available in buildings. If the pupae are found on the body, it is then assumed that either there are obstructions that may have caused some restrictions to larval movement, or a particular species of insect is indicated. Pupae change color from white to dark brown over a specific period, hence, all pupae of whatever color should be collected. A well-planned search strategy should be employed to do the collection. The ideal is to thoroughly search every grid of a meter apart over a 40 square meter area surrounding the decomposing body especially if it is not in a house. This is a gentle and time-consuming activity in which the soil should be sampled at the intercepts of the grid, using a hand trowel to a depth of about 8-10 cm. The soil may need to be sieved over a tray or white cardboard sheet, or it can be hand-searched. As earlier specified, the pupae recovered are placed in a container with a moist paper towel and properly labeled. The pupae do not require feeding but should be taken back to the laboratory for identification. They should be cultured in laboratory conditions until their emergence to ascertain the identification of their species. The pupal cases should also be retained as added evidence. Pupae that fail to hatch provide examples of preserved specimens from the crime scene. Sampling adult flying insects at the crime scene Flying insects found at the crime scene should be collected first with a sweep net, before collecting other specimens probably crawling on or around the body. This is because they can only be captured with a net as they tend to disappear when the scene is disturbed. The net is flicked from behind the insect and lifted up or simply moved up in a sweeping motion with an instant wrist swing so that the net opening could be held up to keep the insects inside it. At this point, the bag can be grasped with the other hand and the insect in the net base can be restricted so that a container can be placed over it. A firm shake usually retains the insect in the bottom of the tube for sufficient time to put a lid on top. 37 | FORENSIC ENTOMOLOGY These insects can either be retained in individual killing jars, or they can be kept until death in a single killing jar as a collection of flying insects from the crime scene. Later, they can be transferred to individual specimen jars. Because insects are mobile, they can be the representative of the crime scene as a whole. In all cases, correct labeling and recording are essential. If the crime scene is a car, for instance, important evidence can be obtained by collecting any insects which have been trapped in a radiator grill, inside the bonnet, or on the windscreen of the vehicle. This may provide trails or details of the movement of the body. The temperatures in the car may be imperative as the inner side of the vehicle is possible to get fairly hot, which may affect the speed of the insect development, as flying insects have gained access and lay eggs. Sampling adult crawling insects at the crime scene Insects such as beetles are visible on the surface of the body. They can be collected by hand-picking and placed individually in a labeled container. This is a functional precaution because beetles may be carnivores and may eat other specimens, thereby destroying your evidence. In an indoor crime scene, it is beneficial to check the corners and crevices of the room for crawling insects as this provides additional information about predators and the conditions in which the body has been found. Leaf litter or ground cover, in an outdoor scene, can also be collected at regular intervals and the contents can be sieved. Pitfall traps can also serve for catching crawling insects near the body in an outdoor crime scene. Another method of collecting crawling insects is the use of Tullgren funnels which can be used to recover the soil organisms living under the decomposing body. Several samples of soil (around 5-8 g each) are ideal. Each is placed in a Tullgren funnel and a light is positioned above the soil sample. As the soil dries out, the organisms are compelled down into the container below containing 70% alcohol. These specimens can later be identified to give a profile of the specimens below the body and elsewhere at the crime scene. Collecting meteorological data at the crime scene It is very important to determine the temperature at which the insects were growing on the dead body before it was discovered. The estimates of time since death depend on the figures gathered at the crime scene and those obtained subsequently from other sources. FORENSIC ENTOMOLOGY| 38 The body temperature of the dead body should be determined by placing a thermometer on the body’s surface. The ambient temperature should be taken at a height of about 1.1 meters or 4 feet (chest height). This offers a measure of air temperature at a comparable height to those taken at the meteorological station. Caution should be taken to avoid holding the actual thermometer, and therefore a protector or a rubber band should be wound around the end. Do not expose the thermometer to direct sunlight, because this may increase the temperature and gives a false reading. Take ambient temperature under a shade. In addition, the temperature directly beneath the body and the soil temperature should be taken successively. It is wiser to use a soil thermometer to take the temperature of the soil so that there is little chance of the thermometer breaking as it is forced into the ground. Rearing Entomological Specimens Recovered from the Crime Scene The larvae of flies and beetles recovered from the crime scene should be reared to the adult stage in laboratory conditions. This should be undertaken by the forensic entomologist so that the identity of the species of insect can be ascertained. Rearing the specimens from the egg stage to the adult, under conditions that simulate the crime scene allows an accurate post- mortem interval to be estimated (Abajue et al., 2017). As each life stage is reached in the laboratory, samples developing from those recovered from each location on the body at the crime scene should be shipped in boiling water and preserved in Kahle’s solution. Data relating to details of temperature and time to reach this stage should also be recorded both in a laboratory note book and on the sample pots so that the pots collected at the crime scene can be related to this information. The record of the data, along with the specimens, may be requested by the court, or be used in court to illustrate your methodology (Gennard, 2007). Insects recovered from a dead body at a crime scene for rearing must be kept in conditions that enable them to grow successfully. Although all of the insects feed on dead bodies, it is not appropriate from both the health and safety standpoints or in terms of human tissue retention laws, to utilize flesh from a corpse to feed insect specimens in the laboratory. A supply of food like a pig’s liver will provide a better alternative to be used at the crime scene to provide food for the samples of living larvae collected from each site on the body. The pig’s liver should also be the food used for the rest of the rearing 39 | FORENSIC ENTOMOLOGY period. Taking entomological evidence to the laboratory The conditions under which the insects are kept are particularly important. Insect specimens must always be kept in proper conditions at the laboratory so that they will be stored without damage or reared through their life stages. The larvae recovered from each of the locations on the body should be separated from each other and stored in separate containers. The likely influence of the temperatures when the specimens are being transported to the laboratory should always be taken into account. If the insects have been collected by a crime scene investigator, they should be grouped according to their life stages. The larvae especially that of flies collected from each of the locations on the dead body should be separately taken to the laboratory from the crime scene at a temperature below the base temperature for those insects expected to be found at the crime scene and handed over to the forensic entomologist as fast as possible. The temperature may need to be determined for local conditions. Myskowiak and Doums (2002) stated that a temperature as low as the normal refrigeration temperature of 4°C may cause alteration in the duration of life stages and the time taken to reach the adult stage. They stated that 10 days of refrigeration before the rearing conditions led to an alteration of 9-56 hours from the duration of 15.5 days counted under normal circumstances where the larvae of Protophormia terraenovae were reared at 24°C. Hence, transit temperatures should be taken into account, mainly if there is any deviation from the expected life cycle in the laboratory. Conditions for fly-rearing in the laboratory When larvae are recovered from the crime scene in foil packages of meat, each package containing about 30 larvae will be placed in containers such as polystyrene cups. These containers can be stored in controlled and dark environments, such as a cabinet or a room, until the larvae reach the post-feeding stage. A pierced lid is placed on the top of each container to reduce drying out; one per sample from each location, for each type of larvae collected. The pierced lid will aid some gas exchange and stop the build-up of ammonia as the larvae grow. These containers should be maintained at a relative humidity between 60 and 65 % or in baths of water at a suitable temperature so that the microclimate within the containers can prevent the eggs or initial larval instars from drying out. Introna et al. (1989) revealed that FORENSIC ENTOMOLOGY| 40 larvae of flies reared in growth cabinets under conditions reminiscent of the wild do not statistically go through alterations in the durations of their normal life cycle. In this instance, they used Lucilia sericata for the trials. As a precaution against loss and intermixing of cultures, each container can be placed in a second container such as an aquarium. Each container should be specifically labeled with the date, case, collector, and item number. For certainty, some adult flies should be kept in large cages to let them mate and to obtain another batch of eggs for development through to the stage they were first recovered at the crime scene. The cages should be around 40 x 30 x 40 cm with a mesh cover to allow light to enter and air to circulate while retaining the culture. If the cage is too small, the insects’ wings will become battered and the flight will be affected, hence mating may or will not take place successfully. Access to the cage is necessary to replace the food through a sleeve opening in the front panel of the cage. The adults are provided with a supply of water continually either through a screw-top jar with a wick emanating the water inside it from the top, a Petri dish with water and stones, or a sponge impregnated with water all of which prevents the flies from drowning while drinking water. The nutritional requirement of the adults is a 50:50 mix of sugar and water to retain the culture (if sugar is used alone without water, the insects may potentially become reduced in size. However, artificial diets have been successfully used to rear blowfly larvae). Meat or liver is also placed in the cage both as nutrients for ovary development in the females and as a place where the eggs are laid. The meat can be grounded or can be palm-sized pieces of liver. While pig liver has been used most successfully, ox or sheep liver can also be used but care should be taken to be consistent with the type and source of meat used to avoid introducing unnecessary variables. Kaneshrajah and Turner (2004) reported that there was a reduction in the rate of larval growth of C. vicina on the liver, in contrast to the growth observed when heart, lungs, kidney, or brain tissues were given as nutrients. However, the majority of researchers successfully use ad libitum liver as the food source for carrion feeders from forensic sites without causing any impact on the duration of life cycle stages. Hermes (1928), however, proved that an effect on the sex ratio of Lucilia sericata was caused by the amount of food available to the larvae, so ad libitum feeding is imperative. The food should be placed in a foil case, or a tub with a partial lid so that the area of access at the top can be reduced. This restriction stimulates flies to lay eggs and lets the 41 | FORENSIC ENTOMOLOGY food retain its moisture for a longer period. Care should be taken to keep the relative humidity above 50% and ideally, 65 % to prevent the eggs from drying out. Flies should be reared at the most suitable temperature, either at the one measured at the crime scene or at the one that allows for swift development. This can be better achieved with a controlled environment cabinet although a room with a temperature with a limited and recorded fluctuation can also be used. Preliminary research indicates a suitable temperature for this end. Information about the expected duration of the life stages comes from several sources including Kamal (1958) who studied the life cycles of 13 fly species at 26.7°C and 50% relative humidity. Anderson (2000), Byrd and Castner (2001), Higley and Haskell (2001), Greenberg and Kunich (2002), and Donovan et al. (2006) are among the others reporting a suitable temperature. The amount of exposure to daylight and dark during a day length which has been successfully used to avoid influencing the life cycle was calculated as 16 hours of daylight and 8 hours of dark (16L:8D; Vaz Nunes and Saunders, 1989). Nevertheless, the most suitable day length to use is the average day length for the season in which the specimens were recovered from the crime scene so that the conditions in the environment before the recovery of the body are mirrored. Conditions necessary for successful rearing of flies to the adult stage Whenever the fly larvae have reached the third instar stage, they must be transferred into conditions that ensure that the post-feeding larvae can migrate successfully, while averting loss of the evidence. The ideal is an aquarium with vermiculite, sand, or sawdust in the bottom, kept in a controlled environment cabinet at the same temperature as that found at the crime scene. This offers the larvae a suitable room for burying themselves to pupate. Therefore, placing the third instar larvae in an aquarium tank or alternative container at this stage of their life cycle if the aquarium has not previously been used as a means of secondary containment. The existence of some extra space is important in that it can affect the degree of success in the completion of the life cycle. Byrd and Castner (2001) noted that the time spent by the larvae in a particular stage could be extended and the PMI estimation will be inaccurate as a result if natural pupation is altered or prevented. FORENSIC ENTOMOLOGY| 42 Rearing beetles in the laboratory Rearing beetles such as Cleridae, Histeridae, Silphidae, and Staphylinidae requires keeping them in transparent plastic containers, glass jars, vials, pots, or buckets. They may need to be kept individually to prevent one beetle from eating another. The containers should have a layer of moist peat, soil, or sawdust in the bottom, depending upon the family of beetles, and places in which insects can hide, such as half-plant pots. Silphid beetles need a temperature of about 20°C and a daylight regime of 16:8 (L:D) (Eggert et al., 1998). They should be kept either individually or in a maximum group size of six beetles of the same sex. Pairs can be placed for breeding in a container including a small carcass such as a defrosted mouse. A large piece of beef, pork, or chicken can aslo offer enough food (Byrd and Castner, 2001). If one is keeping Nicrophorus beetles, a piece of meat left in a container placed on the top of peat will be buried and eggs will be laid in the soil around that piece of meat (Kramer Wilson, 1999). Such eggs can be recovered and retained on moist filter paper at 20°C until they hatch. Eggs of silphid species such as Nicrophorus vespilloides need an average 56 hours to hatch at 20°C (Müller and Eggert, 1990; in Eggert et.al., 1998). Water in a Petri-dish with damp cotton wool or a vial containing water with a wick of paper or cotton wool pushed through the lid should be supplied in the tank for both adult beetles and larvae. The vial is sunk into the peat to let the beetles easily have access to it. The larval culture, each in individual containers, can be supplied with a carcass with a hole in it so that a larva can penetrate it. The cultures can be maintained under a regime of total darkness in an incubator or controlled environment cabinet. Other beetles like the dermestids, do not require a whole carcass to successfully complete their life cycle and can be reared adequately on dried meat or an artificial diet. Dermestids breed optimally at a marginally higher temperature than 20°C and are ideally kept at 25°C and 80% relative humidity (Coombs, 1978). They can also be kept as cultures in aquaria or glass jars. Dermestids need wood sawdust, or some solid medium such as polystyrene or cork where they can burrow to pupate. This should be covered with several layers of paper to simulate conditions in the body. A supply of water must be provided, either as a piece of folded and dampened paper or as a container of 43 | FORENSIC ENTOMOLOGY water with a wick. Keep the water away from the food to prevent fungal growth. Black paper, in the form of a ‘concertina’, provides an egg-laying site from which it is quite simple to see and recover eggs. Nutritive requirements of insects reared in the laboratory Nutritional requirements of both flies and beetles are germane in that they have specific nutrient requirements to be able to complete their life cycles. Flies need carbohydrates as an energy source, water, and protein. Protein is specifically needed by the females for the development of the ovarioles and egg production. They as well require several vitamins and minerals. Beetles associated with carcasses require a diet that integrates the important nutrients. It is suggested that they should be provided with dead mealworms as food every 3-4 days (Eggert et al., 1998). This is the proper food for ground beetles like carabids which can also be fed on ant eggs such as those used for feeding fish, or maggots and pupae. This inclusion of insects in the diet is particularly appropriate for the Cleridae and Silphidae. Others such as Histeridae and Nitidulidae can also be fed in the same way. Conversely, larvae and adults of skin beetles (Dermestidae) - Dermestes maculatus or Dermestes lardarius can be fed on dried dog food or fish food. They prefer pelleted food rather than flakes but can consume both. Such food should be checked every 2 or 3 days to ensure that it is sufficiently consumed and of good quality. The food should be available in excess so that the dermestid life cycle is not influenced (Gennard, 2007). To be sure that dermestids can breed successfully, it may be necessary to provide them with meat on an intermittent basis for the adequate supply of all of the required nutrients for reproduction. To simulate the conditions at the crime scene, using meat such as pork that has dried out may be a more proper food source than artificial foods. Preserving and mounting insect specimens This activity equips an entomologist with the skills that would be required for presenting entomological evidence in court. One will need to mount both adult fly and beetle to provide evidence of the identification features for the species. FORENSIC ENTOMOLOGY| 44 Mounting insects is imperative because it offers examples of insects of different species which can be used for illustrative purposes in the court as well as providing a voucher collection of specimens regarding a particular geographical location. This will allow an entomologist to ascertain whether there are any unusual species present and thus predict the possible movement of a cadaver. The adult insects at the crime scene may have been killed by placing them in a killing jar containing ethyl acetate or any other killing agent. A new jar should be used for each location on the corpse at which the insects were collected. Those from the general crime scene may all be added to the one jar. Note: Ethyl acetate does not cause the insects to become rigid when they die; this means they are easy to position for pinning. Gennard (2007) outlined the following steps in mounting insects: 1. Each large insect, like a blowfly, is removed from the killing jar with the help of fine forceps or a fine paint brush. This is necessary to position it on a cork or polystyrene tile, or insect mounting block. If a mounting block is to be used, choose one large enough on which to sit the thorax so that the insect’s wings and legs can be stretched out. 2. Using a pair of entomological forceps, a pin should be carefully forced through the insect exoskeleton. The pin should be in the size of No. 1 and ideally of a non-rusting type. ▪ For beetles, the pin is placed through the right elytron, as near as possible to the top margin of the wing case. ▪ Large fly specimens should have the pin placed in the middle of their thorax (mesothorax region), exactly between the wings. Smaller flies may be glued to a card. 3. The legs of both flies and beetles should be positioned in a way that enables stretching them away from the body. For example, the first pair of legs of a beetle should be positioned forward, the middle pair of legs should be positioned at a 90° angle to the body and the third pair of legs should be positioned away from the body at a 45° angle to the body. They may need to be held in that position onto the cork or polystyrene, with their wings and legs retained by 45 | FORENSIC ENTOMOLOGY small strips of paper with their ends pinned down. Alternatively, the fly or beetle can be positioned as naturally as possible so that the femur is held in this position and the legs hang down at an angle of 90°. The specimens are left to dry for several days until they are firmly in position. Smaller flies and beetles are less easy to position. They are mounted on a triangular point by gluing. The triangle is usually 1 cm in length and made of white card. Clear nail varnish or Solvate wallpaper paste can both provide a cheap and accessible source of glue. The side of the fly or beetle is glued onto the point of the triangle. The procedure for pinning the specimen and positioning the labels made of white cards is the same. This is irrespective of whether the insect is mounted onto a pin directly, or the triangle plus insect is mounted at the mid-point along its base. To explain how to do this, the pinning and labeling of a fly are described as follows: 4. A pinning block is used to position the fly about 1 cm from the head of the pin. Below this, two labels of an appropriate size for the information and the fly are positioned parallel. 5. First, the pin point is pushed into the fly so that it pierces the thorax. The fly is then moved up the pin into position, by locating the pin point into the hole in the highest step of the pinning block and pushing. 6. Next, the fly is removed from the hole in the top step and the first label is placed on the second highest step. The fly plus pin is pushed into the center of the end of the label so that the information can be read. The label should contain the following information: ▪ Name of the collector. ▪ Place of collection (crime scene), including where on the body. ▪ Item number and case number. ▪ Date collected. 7. The label is pierced by pushing the pin through the label, using entomological forceps and resistance from the block. Once the pin has emerged through the card label, it is slid up the pin to the correct position. This is achieved by pushing the pin down through FORENSIC ENTOMOLOGY| 46 the second hole on the pinning block. It is in a position when the pin hits the bottom of the block. 8. A second label indicates the species name of the insect, when it is identified, and who did the identification. This label is put in the proper position using the same sequence of movements but using the third hole in the lowest step of the pinning block. Both labels are held parallel to each other and written in indelible ink or printed using a laser printer. By using this sequence for mounting insects, the danger of losing information about the case number and location is lessened. The insect can, after all, be re-identified in the unlikely case of loss of the second label on the pinned specimen. Estimating the Post-Mortem Interval of a Corpse After collecting and identifying the insects from the body, the next stage is to relate the information to the temperature at the crime scene. Temperature data including the period since the person was last seen alive are usually obtained from the local meteo¬rological station. These data are ‘corrected’ using a correction factor calculated from the meteorological office data and half-hourly temperature readings which have been recorded at the crime scene for 3-5 days after the body was discovered. These corrected data helps to estimate the temperatures at the crime scene before the corpse was found. Entomologists, using this information, can determine the length of time the flies took to grow from an egg to the developmental stage recovered from the body. By inference, this is the best estimate of the post-mortem interval (PMI) that is available. These estimations of time since death are centered on the speed of insect growth. Insects are ‘cold-blooded’ animals; hence, their growth is influenced by temperature. Below a temperature threshold, development stops; above a specific temperature threshold, the rate of growth also slows down. Between these two points, however, the rate of growth of the juvenile insect is considered to have a linear relationship with ambient temperature. Upper threshold temperatures are not often experienced in investigating most crime scenes, so this factor is not always important. However, if temperatures do remain at or near the maximum for an extended period of time, this will affect the accuracy of the PMI estimate, as the growth of the insect will be slower than expected. Equally, at a very low temperature, development may not take place at all. It is pertinent to note that such 47 | FORENSIC ENTOMOLOGY temperature fluctuations have not been observed in the tropics (personal communication). The temperature threshold below which growth and development will not take place is referred to as the base temperature. This will vary from species to species and with geographic locations. Thus, Davies and Ratcliffe (1994) demonstrated a threshold of 3.5°C for C. vicina in the north of England while Marchenko (2001), working in Russia, recorded a base temperature of 2°C for the same species. Donovan et al. (2006) reported that the base temperature of C. vicina grown in London, at temperatures between 4°C and 30°C, was 1°C. Ther

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