Microscopy and forensic entomology

Abstract

Forensic Entomology is a speciality contributing more frequently to Forensic Sciences. It can be critical for solving forensic cases such as criminal cases through estimation of the postmortem interval, whether the corpse has been moved... or civil cases such as infestation of food or structures, urban pests... In any case, a clear identification of evidence is necessary since a proper expert report depends on the precise knowledge of the species that were involved in each case. It is known that the specific identification of the insects is a complex task, even if they are large, since certain parts of the body, usually of small size, are used. Therefore, from the beginning of Entomology it has been necessary to use microscopical techniques. Most of the studies use optical microscopy, although lately scanning electron microscopy (SEM) is becoming a very popular technique. One of the main goals of Forensic Entomology, the estimation of postmortem interval, deals with the larval stages of Diptera, which need to be identified. Morphological studies are being conducted using optical and electronic microscopy to establish consistent characters of larval stages that will be useful for quick identification, and detemine whether the distinctive characters set can be properly observed at optical microscopy, since this would be a faster way to yield timely results for forensic purposes.

Full text

Microscopy and forensic entomology
N. Ubero-Pascal, I. Arnaldos, R. López-Esclapez and M.D. García
Department of Zoology and Physical Anthropology, University of Murcia, Campus Espinardo s/n. 30100 Murcia, Spaim
Forensic Entomology is a speciality contributing more frequently to Forensic Sciences. It can be critical for solving
forensic cases such as criminal cases through estimation of the postmortem interval, whether the corpse has been moved...
or civil cases such as infestation of food or structures, urban pests... In any case, a clear identification of evidence is
necessary since a proper expert report depends on the precise knowledge of the species that were involved in each case. It
is known that the specific identification of the insects is a complex task, even if they are large, since certain parts of the
body, usually of small size, are used. Therefore, from the beginning of Entomology it has been necessary to use
microscopical techniques. Most of the studies use optical microscopy, although lately scanning electron microscopy
(SEM) is becoming a very popular technique. One of the main goals of Forensic Entomology, the estimation of
postmortem interval, deals with the larval stages of Diptera, which need to be identified. Morphological studies are being
conducted using optical and electronic microscopy to establish consistent characters of larval stages that will be useful for
quick identification, and detemine whether the distinctive characters set can be properly observed at optical microscopy,
since this would be a faster way to yield timely results for forensic purposes.
Keywords SEM; Light Microscopy; Entomology; Forensic Sciences
1. Introduction
Forensic Sciences, defined as the application of scientific methods to legal issues, are an exciting field of study,
complex and multidisciplinary. They use scientific arguments (chemical, physical, biological ...), as well as social and
legal, to the resolution of legal cases. In fact, the term "forensic" defines any science applied to law [1]. The importance
that Forensic Sciences have in the administration of justice resides in its potential ability to provide vital information
about how a criminal action has been committed and who has committed it [2]. Nonetheless, Forensic Science may be
involved in many other activities which, while having legal implications, do not fall within the criminal field. There are
many ways in which Forensic Sciences can be applied, from the estimation of postmortem interval on the basis of
pollen or algae in the corpse to the technology for vocal identification or analysis. The application of Entomology to
legal cases is called Forensic Entomology [1]. Following Hall and Huntington [3], “...is the broad field where arthropod
science and the judicial system interact”. Therefore, Forensic Entomology deals with very diverse applications, the
most well-known being, probably, the medico-legal practice. Although the estimation of postmortem interval on the
basis of entomological evidence is, even, fashionable, Forensic Entomology can also be applied to the evaluation of the
place where death took place, the perimortem circumstances, a guilty or suspicious identification, neglect cases, sudden
deaths, myiasis therapy, detection of drugs or toxins, wild fauna cases, stored products cases, urban and house pests...
All these applications are based on the best possible correct identification of the evidence. Thus, Taxonomy becomes an
essential tool for Forensic Entomology, and Taxonomy is based, in most cases, in morphological characters.
Almost any arthropod can be involved in a Forensic Entomology case. However, the most frequent group consists of
flies (Insecta, Diptera). They are typically related to corpses in the first moments after death, and stay in the corpse for a
variable time depending on the species, the season, the geographical area, etc. Adults, mainly the females, arrive very
early to the corpse to lay eggs (or larvae, in Sarcophagids). After hatching, larvae begin to feed on the corpse tissues.
The development of arthropods is strongly influenced by environmental conditions, mainly temperature. Each species
of fly has different environmental requirements which contribute to its development. The larval age, expressed in hours
or days, is one of the criteria that is used for estimating the postmortem interval (PMI); the estimated age of an
immature insect that has fed on a body provides a minimum PMI [4]. Accurate identification of specimens is usually the
first priority in a forensic analysis of the entomological evidence. Identification of the genus or species allows the
forensic entomologist to access the correct developmental data and distribution ranges to be applied to the case [5]. An
incorrect species determination can lead to a potential error in the estimation of PMI since the carrion species differ in
terms of growth rate, arrival time and position within the order of succession [4].
As mentioned above, the morphological characters are the most important ones for quickly identifying a taxon.
Despite the large number of molecular identification techniques, their cost hinders its implementation. A drawback is
the lost of the specimen studied of which, at best, only fragments can be preserved. On the contrary, morphological
examinations can be done quickly and are extremely cost-effective [5]. For forensic purposes Diptera is the most
interesting group. Among them, three families are most relevant to forensic practice: Calliphoridae (blow flies),
Sarcophagidae (flesh flies) and Muscidae (house and muscid flies) and their larvae are very interesting to estimate the
PMI. These larval stages are not always easily identified and even the morphology of the immature stages of some
species is still unknown. Because of the size, especially in the early stages of development, and the fine details to be
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considered, it is necessary to do a microscopic study. In fact, only few species can be easily identified without the aid of
microscopic study. Scanning electron microscopies (SEM) provides excellent and detailed morphological overview, but
has the disadvantage of being rather expensive and require preparation of the material to be studied. Therefore, we try to
determine the utility of optical microscopy studies by comparing the characters observed with this method and with
SEM in order to determine their applicability to forensic practice.
Fig. 1 Macroscopical morphology of immature stages of C. vicina. a) Comparative size and general morphology of egg and larva
instars. b) General morphology of pupa. c) Pseudocephalon and first thoracic segment of larval instar III. d) General morphology of
anal division of instar III. Magnification: 8x a) and b); 55x c) and d).
2. Materials and methods
The preimaginal stages studied came from colonies bred in laboratory under controlled environmental conditions (25°C
and 60% relative humidity), using pig liver as substrates for egg laying and larval food. Pupation substrate was sand
surrounding the liver. Adults of Calliphoridae were collected in the Campus of Murcia University (Southeastern Spain)
using a modified Schoenly trap [6], and those of Sarcophagidae in Sierra Espuña (Murcia), using the same trap. Muscid
adults were collected in the city of Murcia using pet’s food as bait. All studied specimens, as well as adults, are kept at
the Área de Zoología in the Departamento de Zoología of Universidad de Murcia.
For the stereomicroscope study, larvae were killed in water near to boil and then preserved in 70% ethanol.
Observations were made by a Leica stereomicroscope MZ 9,5 attached to a Nikon digital camera DS-Fi1.
For light microscopy analysis, larvae were killed and preserved in the same way. Afterwards, they were “cleared” so
that the detailed structure of the mouthparts and spiracles (anterior and posterior) coud be studied. This was achieved by
macerating the specimen in a 10% aqueous solution of KOH [7]. Maceration time varies depending on the larval size,
from a few hours for the smallest one to almost one day for to the most developed larvae. Once cleared, larvae were
rinsed and transferred to glacial acetic acid for at least 15 minutes to neutralise any residual potassium [7]. Then,
specimens were mounted on slides to be studied using Hoyer medium. Observations were made using a Nikon 80i
microscope with differential interference contrast (DIC) and pictures were taken using the same camera mentioned
above.
For SEM, selected specimens were rinsed and killed in the usual way (except pupae), and fixed in McDowell fixative
solution, pH 7.4, at 4°C for 24 h. Specimens were rinsed again twice in sodyum cacodylate-sacarose solution;
dehydrated until absolute acetone in a gradient of increasing concentration of ethanol and ethanol-acetone solution
(50% of each reagent), and either dried using critical point method or air-dried by hexamethyldisilizane treatment [8].
Dried specimens were mounted on stub with conductive adhesive tape, trying to lay them in different position (dorsal,
lateral and ventral views). Because the third instar larvae are the largest ones, they were cut in order to study efficiently
the fore and hind parts of the body. Specimens were coated with Au-Pd in a Polaron Bio Rad Sputter Coat and observed
in a JEOL JSM 6100 SEM located in the Microscopy Service of Murcia University. Pictures were directly digitized
from the SEM.
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The considered morphological features of eggs, larvae and pupae, the body divisions and the terminology follow
those of [9, 10, 11, 12, 13].
Abbreviations used in figures: a, antenna; ae, anterior spiracle; an or ap, anal protuberance; b, button; bm, bubble
membrane; c, chorion; cl, cephalic lobe; eb, spines band; h, respiratory horn; la, labial lobe; m, micropyle; ma, median
area; mp, maxillary palpus; og, oral groove; p, papillae; pa, anterior pole; pe, posterior spiracle; pl, respiratory plastron;
pp, posterior pole; pr, peritreme; ps, pseudocephalon; psc, pseudocephalon collapsed; pt, peristigmatic tuft; rs,
respiratory slit; sk, cephaloskeleton; vo, ventral organ.
Fig. 2 General morphology and ultrastructure of eggs in C. vicina (a-b, d, f) and S. nudiseta (c, e, f-g). a) General morphology by
light microscopy. b and c) General morphology by SEM. d and e) Ultrastructure of anterior pole and micropyle. f and g)
Ultrastructure of plastron area. Magnification light microscopy: 30x a).
3. Results and Discussion
Eggs, three larval instars and pupae are the immature stages in a typical Diptera life cycle (Fig. 1 a-b). The morphology
of all these stages provides many data of great interest for taxonomic purposes. However, many Diptera species of
forensic importance are still not well known from a morphological point of view, or the main study available for a
specific identification was only performed with light microscopy. This kind of microscopy can be useful for a general
overview of immature stages (Figs. 1 and 2a), the study of big structures like band of spines or anterior and posterior
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spiracles (Fig. 4c-d) in specimens of great size (instar III of larvae and pupae) and, even, for studying internal structures
such as mouthparts or cephalopharingeal skeleton (Fig. 3a-b). However, the morphology of stages whose specimens are
small, such as eggs (Fig. 2a) and larval instars I and II, or tiny structures in the other stages (Fig. 3e), are poorly
described. This implies two taxonomical problems. One relates to the fact that general description by light microscopy
only allows to distinguish species very different morphologically and only in the last stages of life cycle (instar III and
pupae). Second, light microscopy does not allow connecting first and late larval stages if they are not bred in the lab,
because this technique doesn’t allow a good discrimination of morphological characters of taxonomic importance. So,
SEM is now being used for this purpose, providing fine details of morphological features that allow distinguishing
species regardless of stage and connecting the stages without having to breed them in the lab. Nevertheless, the
morphological knowledge of immature stages of Diptera species of forensic importance using SEM is currently
insufficient to establish the best characters to accomplish this task.
Below, we will detail the usefulness of microscopy for the study of Dipteran immature stages, illustrating what each
technique affords and how they can complement each other. The purpose is to define the key features at SEM that could
also be identified using light microscopy, since this technique is easier to use, and results can be obtained more quickly.
The fast processing feature is crucial for solving forensic cases in a brief period of time.
Fig. 3 Internal and external morphological features of pseudocephalon in Phaenicia sericata (Meigen, 1826) (a), S. nudiseta (b, d,
e), Sarcophaga cf. cultellata Pandelle, 1896 (c) and C. vicina (f). a and b) Cephaloskeleton morphology by light microscopy. c and d)
Ultratructure of pseudocephalon. e) Morphology of antenna and maxillary palpus by light microscopy. f) Ultrastructure of antenna
and maxillary palpus. Magnification light microscopy: 40x a and b); 400x e).
3.1 Morphology of eggs
A
ccording to Smith [14], eggs of Diptera of forensic importance are very simple morphologically although some main
or typical structures can be distinguished [10, 15]: micropilar plate, median area, respiratory plastron, hatching lines,
and chorion sculpturing. Diptera eggs are larger than those already studied in other groups, such as Ephemeroptera [16],
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however SEM is necessary for a detailed morphological analysis [10]. Light microscopy doesn’t provide the accurate
observation necessary to describe the main chorionic structures (Fig. 2a). In fact, all previous papers that use eggs to
distinguish Diptera species are performed by SEM [10, 17, 18, 19]. Nevertheless, not all Diptera especies of forensic
importance can be distinguished by the egg morphology, since they are very similar, and specific features have not been
yet described. This is the case of species belonging to the same genus, such as Calliphora [10]. Despite it, egg
morphology allows to distinguish many species of Diptera. An example is showed in Fig. 2b-g, where it can be seen
that eggs of Calliphora vicina Robineau-Desvoidy, 1830 and Synthesiomya nudiseta van der Wulp, 1883 are very
similar in their general shape or in the location of micopyles, but they also show clear differences. For instance: 1)
plastron runs along the complete length of egg in S. nudiseta, while in C. vicina doesn’t arrive to the posterior pole (Fig.
2 b-c), having clear morphological differences (Fig. 2f-g); 2) boundaries of median area are expanded in S. nudiseta,
while in C. vicina are very short (Fig. 2b-e); and c) chorionic sculpturing is clearly different, while S. nudiseta shows
longitudinal ribs and a great magnification is necessary to observe the hexagonal chorionic reticulation (Fig. 2e), C.
vicina has not ribs and the hexagonal reticulation is observed at very low magnification (Fig. 2d). In this respect, many
structural variations have been described among eggs of other species, mainly in the plastron structure and the
boundaries of median area [10, 15, 17, 18, 19].
Fig. 4 Morphological features of anal division in S. nudiseta (a, c, e), S. cf. cultellata (b), P sericata (d) and C. vicina (f). a and b)
Ultrastructure of anal division. c and d) Morphology of posterior spiracle by light microscopy. e and f) Ultrastructure of posterior
spiracle by SEM. Magnification: 200x c and d).
It is noteworthy that in some Diptera of forensic importance eggs can not be studied because the species are
ovoviviparous and lay first stage larva directly, as normally happens with Sarcophagidae and, also, with some
Calliphoridae when food is scarce [14].
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Fig. 5 Morphological comparison of pseudocephalon and anal division in the three larval instars of C. vicina. a and b) Instar I. c and
d) Instar II. e and f) Instar III.
3.2 Morphology of larvae
Larvae are the main source of morphological useful data to identify Diptera of forensic importance in immature stages,
specially the instar III (Figs. 3 and 4). Although the size of larvae changes drastically from one instar to other, usually
instar III may measure five or six times the length of instar I (Fig. 1a), the morphology of the body is basically
maintained in all of them (Fig. 1a). Following Courtney et al. [11], larvae of cyclorraphan Diptera are composed of
twelve segments. Three regions can be differentiated in the body: cephalic region or pseudocephalon (first segment),
thorax (from second to fourth segments) and abdomen (from fifth to twelfth segments), although the twelfth segment is
also known as anal division. Every region and even each segment shows several unique structures that usually appear in
all instars, although they may appear more complex in the last instar (Fig. 5). These structures allow to distinguish
between species and to relate larval instars in the same species (Figs. 3-5).
External larval morphology provides the main number of structures of taxonomical value, although the mouthparts or
cephalopharingeal skeleton, an internal structure, is widely used for this purpose, especially in instar III [14]. Light
microscopy is the appropriate technique to study the cephalopharingeal skeleton without dissecting the specimen since
all sclerites that form it do not move and maintain their position (Fig. 3a-b). However, to get good observations it is
necessary to clear the larvae. All larval instars are provided of cephalopharingeal skeleton and this structure is useful to
distinguish species in all instars [9, 17]. However, this structure is mainly known for instar III, and there are quite few
species in which this structure has been described for all larval instars. Today, the scientific movement preparing
identification keys for Diptera species of forensic importance in instar III continue to consider the cephalopharingeal
skeleton as a specific structure that is necessary to know.
Light microscopy is also useful to study the external morphology of larvae in all instars but, sometimes, the
resolution of this device is inappropriate for observing in detail some structures (Figs. 3e and 4c-d). SEM has shown
these structures as having a very rich ultrastructure with many morphological details (Figs. 3f and 4e-f) [20]; some of
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them could be species selective. The pseudocephalon and anal division are the larval body segments in which the main
specific structures appear (Fig. 3e-d), while the remaining segments show a smaller number of structures, very
important in some families. Cephalic lobes, antennae, maxillary palpus, ventral organ, oral ridges, labial lobe… are
some of the structures that can be observed in the pseudocephalon of instar III using light microscopy (Fig. 3a-b, e).
However, in order to observe these structures in instars I and II or to describe its detailed morphology it is necessary
their study at SEM (Figs. 3e-d, f and 5a,c). This allows to determine that cephalic lobe shows many sensillae; antenna is
composed of a dome and a basal ring with an external sensilla, maxillary palpus consists of several sensillae located in
different places, ventral organ is a complex structure,… but the most important consideration is that all these structures
can be studied in all instars with a good resolution (Fig. 5a,c).
Anal division has many structures of taxonomic value and some of them can be differentiated macroscopically (Figs.
1d and 4c-d). Posterior spiracles, posterior papillae, anal pad… are some of the structures of taxonomic interest that can
be studied at light microscopy, specially in instar III. However, for describing the respiratory slits of posterior spiracles,
the peristigmatic tufts, the peritrema ornamentation, the sensillae in posterior spiracle, or simply the sculpturing of
tegument, it is necessary to use SEM (Fig. 4a-b, e-f). The description of these structures by SEM allows their correct
interpretation and to establish the proper correlations with the light microscopy observation, for the most possible
accurate identification of species.
The anterior spiracle morphology, spines arrangement in segments and tegument sculpturing are also larval features
of interest to identify species and, although some of them can be observed by light microscopy, SEM provides
exceptional images to be described.
On the other hand, the description of structures by SEM is indispensable for an effective correlation of larval
morphology of one species in its three instars [20, 21, 22, 23]. For instar I the light microscopy is insufficient to
describe the external morphology. Moreover, instar I may present a structure arrangement very different to instars II and
III, which are very similar morphologically (Fig. 5). Several studies have been published which focused in the
description of instar I in many species [13, 24, 25] and the relationship with other larval instars because, sometimes, this
larval stage is the only evidence in a forensic case and, usually, the specimens arrive dead to the researcher, being
impossible their breeding to adults.
Fig. 6 Ultrastructure of pupae of C. vicina. a) Pseudocephalon collapsed. b) Respiratory horn and traces of bubble membrane. c)
General morphology of anal division. d) Detail of morphology of posterior spiracle.
3.3 Morphology of pupae
Usually, the pupa of Diptera of forensic importance shows most of the morphological characters of larval instar III [9,
17], except for some collapsed parts, such as the pseudocephalon (Fig. 6a). The arrangement of spines in segments and
anal division structures are more or less conserved in the pupa (Fig. 6c), specially the posterior spiracles (Fig. 6d),
allowing the correlation to larval morphology for identifying purposes. These structures can be observed by light
microscopy although it is better described by SEM. In fact the main description of pupae has been given by this
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technique. Moreover, pupae morphology shows exclusive structures, such as the bubble membrane and respiratory
horn, which are very close related [9, 17]: bubble membrane appears in young pupae and disappears when the
respiratory horn is developed, since both structures are placed in the same location (Fig. 6b). Although some papers
describe the bubble membrane by light microscopy, according to Liu and Greenberg [17] SEM is the best technique to
describe this membrane because the bubble protuberance may be very tiny. Therefore, for a correct study of pupae both
microscopy techniques are necessary.
4. Conclusions and general remarks
Currently, the description and identification of immature stages of Diptera useful for forensic sciences at the specific
level requires the complementary use of light microscopy and SEM, because each one supplies different information
completely useful for these purposes. For a long time, light microscopy has been the most used technique and continues
to be important, since the procedures to observe the samples are faster than SEM. In fact, the keys developed by some
scientists for the identifications of larvae are based mainly on the body observations by this technique. However these
keys are limited to larval instar III. Although some species can be identified in larval instars I and II by light
microscopy, this technique is very limited for a complete description of their morphology, especially in instar I, and for
finding taxonomical characters in uncharacterized species to allow the correct specific identification. For these reason,
SEM is increasingly being adopted by forensic entomologist as a new device to obtain precise information about larval
body morphology, not only in eggs and instars I and II, but also for recharacterizing instar III and pupae. The joint use
of both microscopical techniques is appropriate for a complete knowledge of larval morphology in all instars. This
circumstance is allowing to establish new specific characters based on SEM observations that can be visualized by light
microscopy for a quick identification of the specimens and, also, to correlate all the immature stages in each species.
However, further efforts are necessary, especially by SEM, to characterize the immature stages in all species of Diptera
of forensic interest.
Acknowledgements Th
is contribution has been supported by the projects 00848/CV/01 of Fundación Séneca of the Comunidad
Autónoma de la Región de Murcia and CGL20205-04668/BOS of Ministerio de Educación y Ciencia of the Spanish Government.
References
[1] Walker M. Entomology and Palynology. Evidence from the Natural World. Broomall, Pennsylvania: Mason Crest Publishers;
2006.
[2] Kiely, T.F. Forensic Evidence. En: James SH, Nordby JJ. Forensic Science. An Introduction to Scientific and Investigative
Techniques. Second Edition. Boca Raton: CRC Press; 2005: 649-666.
[3] Hall RD, Huntington TE. Introduction: perceptions and status of Forensic Entomology. In: Byrd JH, Castner JL, eds. Forensic
Entomology. The utility of arthropods in legal investigations. Boca Raton: CRC Press; 2010: 1-16.
[4] Wells JD, Lamotte LR. Estimating the postmortem interval. In: Byrd JH, Castner JL, eds. Forensic Entomology. The utility of
arthropods in legal investigations. Boca Raton: CRC Press; 2010: 367-388.
[5] Byrd JH, Castner JL. Insects of forensic importance. In: Byrd JH, Castner JL, eds. Forensic Entomology. The utility of
arthropods in legal investigations. Boca Raton: CRC Press; 2010: 39-126.
[6] Arnaldos MI, Romera E, Presa JJ, Luna A, García MD. Studies on seasonal arthropod succession on carrion in the southeastern
Iberian Peninsula. International Journal of Legal Medicine. 2004; 116: 197-205.
[7] Smith KGV. A manual of Forensic Entomology. London: The Trustees of the British Museum (Natural History); 1986.
Ubero-Pascal N, Fortuño JM, Puig MA. New application of air-drying techniques for studying Ephemeroptera and Plecoptera
eggs by Scanning Electron Microscopy. Microscopy Research and Techniques. 2005; 68: 264-271.
[9] Erzinçlioglu YZ. The larvae of the species of Phormia and Boreellus; Northern, cold-adapted blowflies (Diptera:
Calliphoridae). Journal of Natural History. 1985; 22: 11-16.
[10] Erzinçlioglu YZ. The value of chorionic structure and size in the diagnosis of blowfly eggs. Medical and Veterinary
Entomology. 1989; 3: 281-285.
[11] Courtney GW, Sinclair BJ, Meier R. Morphology and terminology of Diptera larvae. In: Papp L, Darvas B, eds. Contribution
to a Manual of Paleartic Diptera: General and Applied Dipterology (Vol 1). Budapest: Science Herald; 2000: 85-161.
[12] Sukontason KL, Piangjai S, Bunchu N, Chaiwong T, Sripakdee D, Boonsriwong W, Vogtsberger RC, Sukontason K. Surface
ultrastructure of the puparia of the blow fly, Lucilia cuprina (Diptera: Calliphoridae), and flesh fly, Liosarcophaga dux
(Diptera: Sarcophagidae). Parasitology Research; 2006: 98: 482-487.
[13] Szpila K, Pape T, Rusinek A. Morphology of the first instar of Calliphora vicina, Phormia regina and Lucilia illustris
(Diptera, Calliphoridae). Medical and Veterinary Entomology. 2008; 22: 16-25
[14] Smith K.G.V. An introduction to the immature stages of British Flies. Diptera larvae, with notes on eggs, puparia and
pupae. Handbooks for the Identification of Britsh Insects, Vol. 10, part 14, Dorset: Royal Entomological Society of London,
Henry Ling Ltd.; 1989.
[15] Hinton N. Biology of Insects eggs. Oxford: Pegamon Press; 1981.
[16] Ubero-Pascal N, Puig MA. Microscopy and egg morphology of Mayflies. In: Méndez-Vilas A, Díaz J, eds. Modern Research
and Educational Topics in Microscopy. Badajoz: Formatex; 2007: 326-335
Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
©FORMATEX 2010
1555
______________________________________________

[17] Liu D, Greemberg B. Immature stages of some flies of forensic importance. Annals of the Entomological Society of America.
1989; 82: 80-93.
[18] Pires de Alencar AP, Rios AC. Ultrastructure of the egg of Muscina stabulans and Synthesiomyia nudiseta (Diptera:
Muscidae). Mem. Inst. Oswaldo Cruz. 1992; 87 (4): 463-466.
[19] Mendoça PM, dos Santos-Mallet JC, de Mello RP, Gomes L, de Carvalho MM. Identification of fly eggs using scanning
electron microscopy for forensic investigations. Micron. 2008; 39 (7): 802-807.
[20] Colwell DD, Otranto, D, Horak IG. Comparative scanning electron microscopy of Gasterophilus third instars. Medical and
Veterinary Entomology. 2007; 21: 255-264.
[21] Sukontason KL, Sukontason K, Lertthamnongtham S, Kuntalue B, Thijuk N, Vogtsberger RC, Olson JK. Surface
ultrastructure of Chrysomya rufifacies (Macquart) larvae (Diptera, Calliphoridae). Journal of Medical Entomology. 2003; 40:
259-267.
[22] Sukontason KL, Sukontason K, Piangjai S, Bunchu N, Chaiwong T, Vogtsberger RC, Kuntalue B, Thijuk N, Olson JK
(2003a) Larval morphology of Chrysomya megacephala (Fabricius) (Diptera, Calliphoridae) using scanning electron
microscopy. Journal of Vector Ecology. 2003; 28, 47-52.
[23] Sukontason KL, Sukontason KL, Piangjai S, Chaiwong T, Boonchu N, Kurahashi H, Vogtsberger RC (2003c) Larval
ultrastructure of Parasarcophaga dux (Thomson) (Diptera: Sarcophagidae). Micron. 2003; 34: 359-364
[24] Szpilla K. First instar larvae of nine West-Paleartic species of Pollenia Robineau-Desvoidy, 1830 (Diptera: Calliphoridae).
Entomologica Fennica. 2003; 14: 193-210.
[25] Szpila K, Pape T. Comparative morphology of the first instar of three species of K Meigen (Diptera: Sarcophagidae,
Miltogramminae). European Journal of Entomology. 2005; 104: 119-137
Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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