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Recognition in burying beetles (Nicrophorus spp., Silphidae, Coleoptera) [Elektronische Ressource] / vorgelegt von Sonia Whitlow

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138 pages
Recognition in burying beetles (Nicrophorus spp., Silphidae, Coleoptera) Inauguraldissertation zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg in Breisgau Vorgelegt von Sonia Whitlow 2003 Dekan der Fakultät für Biologie: Prof. Dr. H. Kleinig Promotionsvorsitzender: Prof. Dr. K.F. Fischbach Betreuer der Arbeit: Prof. Dr. K. Peschke, Prof. Dr. J.K. Müller Referent: Prof. Dr. K. Peschke Koreferent: Prof. Dr. J.K. Müller 3. Prüfer: Prof. Dr. S. Rossel Tag der Verkündung des Prüfungsergebnisses: 5. Februar 2004 Contents Contents Page number 1 Introduction 1 1.1 The insect cuticle 1 1.2 Case study: Nicrophorus species 3 1.3 Questions to be addressed 4 2 Materials and methods 6 2.1 Capture, care and breeding 6 2.2 Preparation and performance of behavioural experiments 6 2.2.1 Washing 6 2.2.2 Behavioural tests 7 2.3 Chemical analyses 8 2.3.1 Extraction 8 2.3.2 Silica gel chromatography 8 2.3.3 Solid-phase microextraction (SPME) 8 2.3.4 Dimethyl disulphide (DMDS) derivatisation 9 2.3.5 Gas chromatography and mass spectrometry (GC and GC-MS) 9 2.3.6 Identification of chemicals 10 2.3.6.1 n-Alkanes 10 2.3.6.2 Olefins 11 2.3.6.
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Recognition in burying beetles
(Nicrophorus spp., Silphidae,
Coleoptera)







Inauguraldissertation
zur Erlangung der Doktorwürde
der Fakultät für Biologie
der Albert-Ludwigs-Universität
Freiburg in Breisgau







Vorgelegt von Sonia Whitlow




2003









Dekan der Fakultät für Biologie: Prof. Dr. H. Kleinig
Promotionsvorsitzender: Prof. Dr. K.F. Fischbach
Betreuer der Arbeit: Prof. Dr. K. Peschke, Prof. Dr. J.K. Müller
Referent: Prof. Dr. K. Peschke
Koreferent: Prof. Dr. J.K. Müller
3. Prüfer: Prof. Dr. S. Rossel

Tag der Verkündung des Prüfungsergebnisses: 5. Februar 2004 Contents
Contents Page number

1 Introduction 1
1.1 The insect cuticle 1
1.2 Case study: Nicrophorus species 3
1.3 Questions to be addressed 4

2 Materials and methods 6
2.1 Capture, care and breeding 6
2.2 Preparation and performance of behavioural experiments 6
2.2.1 Washing 6
2.2.2 Behavioural tests 7
2.3 Chemical analyses 8
2.3.1 Extraction 8
2.3.2 Silica gel chromatography 8
2.3.3 Solid-phase microextraction (SPME) 8
2.3.4 Dimethyl disulphide (DMDS) derivatisation 9
2.3.5 Gas chromatography and mass spectrometry (GC and
GC-MS) 9
2.3.6 Identification of chemicals 10
2.3.6.1 n-Alkanes 10
2.3.6.2 Olefins 11
2.3.6.3 Methyl-branched hydrocarbons 13
2.3.6.4 The position of the double bond 14
2.4 Statistical methods 15

3 Results 18
3.1 Types of chemicals 18
3.1.1 Hydrocarbons 18
3.1.2 n-Alkanes 18
3.1.3 Olefins 18
3.1.4 Monomethylalkanes 19
3.1.5 Dimethylalkanes 19
3.1.6 Trimethylalkanes 20
3.1.7 Other cuticular chemicals 20
3.2 Contamination of the cuticular extract 20
3.2.1 Haemolymph 20
3.2.2 Anal secretion 22
3.2.3 Substrate 22
3.3 Body parts 23
3.4 The pattern of cuticular chemicals 25
3.5 Chain length 25
3.6 The species-specific pattern of chemicals 27
3.6.1 N. vespilloides (E) 27
3.6.2 (C) 29
3.6.3 N. defodiens 29
3.6.4 N. vespillo 30
3.6.5 N. humator 30
3.6.6 N. fossor 31
3.6.7 N. sayi 31
3.6.8 N. orbicollis 31 Contents
3.6.9 N. tomentosus 31
3.6.10 Necrodes littoralis 31
3.6.11 Oiceoptoma thoracica 31
3.6.12 Ptomascopus morio 32
3.7 Species specificity of the cuticular chemicals 32
3.8 Principal Component Analysis (PCA) 39
3.8.1 N. vespilloides (E) 39
3.8.2 (C) 42
3.8.3 N. vespillo 43
3.8.4 N. humator 45
3.8.5 N. defodiens 47
3.9 The recognition system in N. vespilloides (E) 52
3.9.1 Response of females to mature conspecific females 52
3.9.2 Information in the cuticular chemicals used by females 52
3.9.3 The sex-specific pattern detected by females 54
3.9.4 Response of males to mature conspecific females 55
3.9.5 Information in the cuticular chemicals used by males 56
3.9.6 The sex-specific pattern detected by males 57
3.9.7 Response of females to immature conspecific individuals 60
3.9.8 Response of males to immature conspecific individuals 61
3.9.9 Response of N. vespilloides (E) males to other species 63
3.9.10 Response of (E) females to other species 64

4 Discussion 66
4.1 The insect cuticle 66
4.2 Types of hydrocarbons 67
4.2.1 n-Alkanes 67
4.2.2 Olefins 67
4.2.3 Monomethylalkanes 67
4.2.4 Dimethylalkanes 68
4.2.5 Trimethylalkanes 68
4.3 The prevention of desiccation 69
4.4 Species-specificity of the cuticular pattern 70
4.5 Sex-specificity 72
4.6 Age- 73
4.7 The recognition process in Nicrophorus species 73
4.7.1 The importance of cuticular chemicals in recognition 74
4.7.2 Determination of conspecifics by male N. vespilloides (E) 74
4.7.3 Determination of conspecifics by female (E) 75
4.7.4 Determination of immature conspecifics by female
N. vespilloides (E) 76
4.7.5 Determination of immature conspecifics by male
(E) 77
4.7.6 Interactions with different species 77

5 Conclusions 80

6 Bibliography 82

7 Acknowledgements 90
Contents
8 Appendix 91
Introduction - 1 -
1. Introduction
Burying beetles (Nicrophorus species) are unusual in that they have an extensive biparental brood
care system. This is rare in insects and such a complicated system can be found in very few species
e.g. the desert tenebrionid beetle, Parastizopus armaticeps (Rasa 1999). The method by which
males and females are able to distinguish each other as potential sexual partners may be the ability of
individuals to detect sex- and species- specific chemicals or patterns of chemicals on the cuticle.

1.1 The insect cuticle
The waxy (lipid) layer on the cuticles of insects plays a vital role in many aspects of insect life. These
lipids, which cover the surface of the cuticle, consist mainly of hydrocarbons, particularly n-alkanes,
methylalkanes and olefins. Small amounts of fatty acids, fatty acid esters and aldehydes are also
present (Jackson & Blomquist 1976).

The main purpose of the waxy layer is to help to prevent desiccation (Hadley 1984), a function
which has allowed insects to successfully colonise land. Individuals of the same species e.g. the
cockroach, Blatella germanica, are often found to vary the quantity and abundance of certain
hydrocarbons with habitat and season to reduce transpiration as much as possible (Young et al.
2000). Other uses include the prevention of the entry of micro-organisms, and the exploitation of
information as kairomonal cues, which parasites use to find their hosts (Review: Blomquist & Dillwith
1985). The complex nature of the lipids has also allowed insects to evolve a very effective
communication system. The cuticular pattern has been found to enable discrimination of species and
sexual partners, as well as the release of mating behaviour at close range, and nest mate recognition
in social insects, for example ants (Formicidae), honey bees (Apidae) and paper wasps (Vespidae)
(see Singer 1998 for a general review). Termites are another excellent example of social insects
which use cuticular components for recognition systems. The cuticular patterns of each species of
termite are unique (Haverty & Thorne 1989). If an individual of a different species enters a colony,
members of that colony eject the intruder from the nest. It was noted that the signature for this
recognition was present in the hydrocarbon fraction of the cuticular extract which allowed
discrimination of individual specificity (Bagnères et al. 1991).
Introduction - 2 -
The presence of a sex pheromone on females allows males to discriminate conspecific sexual
partners and releases copulatory behaviour. In the rove beetle, Aleochara curtula, two main sex
pheromones were identified, (Z)7-heneicosene and (Z)7-tricosene (Peschke & Metzler 1987). It
was found that immature males also produce the female sex pheromone, but the levels decreased
during the first two weeks after emergence (Peschke 1986). In the house fly, Musca domestica,
females are recognised mainly by the presence of (Z)9-tricosene (Dillwith et al. 1982).
Vitellogenesis stimulates changes in the cuticular hydrocarbon pattern and triggers production of the
sex pheromone (Carlson et al. 1971; Dillwith et al. 1982). The presence of methylalkanes further
increases the copulatory attempts of males (Nelson et al. 1981). Olefins were also found to release
copulatory behaviour in some Drosophila species (Oguma et al. 1992).

Chemicals which just appear on the cuticles of either males or females of a species allow for easy
discrimination. However, many species do not show any qualitative differences in the cuticular
patterns of mature males and females. For example, not all species in the Drosophila virilis group
had female-specific sex pheromones (Bartelt et al. 1986). In spite of this, males still release sexual
behaviour when they came into contact with females and this may be due to certain cuticular
hydrocarbon patterns on the females. This is also the case with alfalfa leaf cutter bees, Megachile
rotundata. It was found that females have higher concentrations of certain n-alkenes than males
(Paulmier et al. 1999). The absence of a unique sex pheromone still allows recognition of
conspecific females by the ability of males to determine differences in the proportions of chemicals on
individuals. In fact, it is possible that the insects may be reacting to threshold levels of one or more
chemicals (Cobb & Jallon 1990).

Age-related differences in the cuticular patterns have been noted in many species (e.g. some
Drosophila species, Bartelt et al. 1986; the queenless ant Diacamma ceylonese, Cuvellier-Hot et
al. 2001; the wasp, Polistes fuscatus, Panek et al. 2001). These differences were found not only in
the production of sex pheromones as the individuals matured, but also in other hydrocarbons. Bartelt
et al. (1986) reported that immature individuals in the Drosophila virilis group had a different
cuticular pattern to the mature individuals. As the flies matured, there was an increase in the relative
abundance of the short-chained hydrocarbons. In Glossina morsitans, young adults had a series of Introduction - 3 -
hydrocarbons with a chain length below C which disappeared during maturation in the first few 29
days of adult life (Huyton et al. 1980).
It is thought that the majority of cuticular chemicals are synthesised by the insects themselves by
elongation and decarboxylation reactions, with only a small fraction coming directly from the diet
(Blomquist & Jackson 1973). The cuticular chemical pattern may be altered slightly by the
environment but the basic pattern is a reflection of the genotype and is potentially useful for taxonomy
(Lockey 1976). The cuticular patterns can be used to determine sub-species, which is especially
useful when there are no morphological differences between them (e.g. Zootermopsis nevadensis
nevadensis and Zootermopsis n. nuttingi, Haverty & Thorne 1989). However, the actual suitability
of cuticular hydrocarbons for chemotaxonomy has yet to be demonstrated. For example, Lockey
(1982a, 1982b, 1982c, 1984), studied adult tenebrionid beetles and found characteristics at the
genus, species and tribe level. Lockey & Metcalfe (1988) analysed data from tenebrionid beetles as
well and built up dendrograms based on hydrocarbon compositions. However, these dendrograms
did not correlate exactly with the established taxonomy. In general, however, relatively few insect
species have been studied and some isomeric mixtures of olefins and methylalkanes have not yet
been correctly resolved by gas chromatography. Thus little can be said about the value of
chemotaxonomy at this stage (Lockey 1991).

1.2 Case study: Nicrophorus species
The subject of the present study is the genus Nicrophorus. These beetles are quasisocial insects
which raise their young on small vertebrate carcasses. Males and females are attracted to carcasses
by olfaction, and a small carcass suitable for use in reproduction tends to be discovered by many
burying beetles of different species within a short space of time. Suitable carcasses are rare and thus
difficult to find, so competition for them is intense. Fights occur intraspecifically with individuals of the
same sex, - males against males and females against females. Interspecific fights occur regardless of
sex (Pukowski 1933; Müller et al. 1991), in which the largest individuals usually win the fights
(Dressel 1987). A pair of conspecific ‘winners’ monopolises the carcass, and raises its young
together on the carcass.

Carcasses are often found by several individuals of different species within a short space of time.
These individuals may include not only those of Nicrophorus species, but also carabids, other Introduction - 4 -
silphids, nitidulids, scarabaeid dung beetles, and some hymenopterans and dipterans (Eggert &
Müller 1987). Nicrophorus individuals are also able to recognise non-Nicrophorus species as
competitors for the carcass (Pukowski 1933). It has been observed that, for example, blow fly eggs
and maggots are destroyed and eaten by burying beetles (Scott 1994). By burying the carcass,
Nicrophorus species manage to hide it, so that insects other than Nicrophorus are less likely to find
it and take it over. Also, burying beetles in general have a large and heavily sclerotized body, which
is effective in fights (Eggert & Müller 1987). Wilson & Fudge (1984) discovered that if a carcass has
been found by several individuals of the same species, intraspecific competition occurs once the
carcass has been buried. It is absolutely essential that Nicrophorus species are able to determine
which individuals are potential partners with which young can be raised, and others which want to
take over the carcass for their own use, i.e. competitors for the carcass.

It is not known exactly how Nicrophorus species are actually able to differentiate each other. As
early as 1933, Pukowski suggested that Nicrophorus species were able to pick up information
about individuals using chemoreception. It was noticed that when two individuals met, there was brief
contact between the antennae and the body of the other individual. It was suggested that substances
present on the surface of the cuticle may carry information about the individual. Due to the ever
increasing evidence that the cuticular chemicals on insects carry essential information, it may also be
the case in burying beetles and this possibility is researched in the present work.

1.3 Questions to be addressed
The aim of this work is to determine whether the cuticular pattern in Nicrophorus species is species,
sex- and age-specific. The question of whether any differences in the cuticular patterns are used by
the individuals themselves to distinguish others, or whether they merely reflect a phylogenetic pattern
is addressed.

The cuticular chemicals were identified by gas chromatography and mass spectrometry (GC-MS)
and were quantitatively analysed by measuring the peak areas of the resulting chromatograms. The
peak areas were used in principal component analyses (PCA) to determine if the chemicals were
sex- and age-specific. This also gave clues as to which chemicals distinguished the groupings. Those Introduction - 5 -
chemicals which were responsible for the differences between mature male and female N.
vespilloides from the European population were then tested for their biological activity in biotests.

Cluster analyses were used to group the species according to the similarities in the cuticular
chemicals. The resulting tree diagrams were compared to a phylogenetic tree diagram to determine
the effectiveness and usefulness of the cuticular chemicals in chemotaxonomy. Individuals of carrion
beetles other than Nicrophorus were also included in this test to discover if there are genus-specific
cues.

Behavioural tests were carried out to discover whether cuticular chemicals were necessary for the
ability of Nicrophorus individuals to discriminate conspecific potential partners from others, using the
European N. vespilloides population as an example. The presence of a carcass in these experiments
increases the readiness of individuals to fight against any individuals other than conspecific potential
partners, i.e. competitors, to gain possession of the carcass. Cuticular extracts or fractions of
extracts were added to either dead individuals or washed (odourless) individuals to determine their
importance in the recognition system of N. vespilloides. Specific synthetic chemicals, determined as
being important in sex-specificity in the results of the PCA analyses, were added to the cuticle of
dead individuals to determine whether they had any biological activity.

The reaction of mature European N. vespilloides towards immature conspecifics was tested on a
carcass to determine their ability to distinguish whether an individual was sexually mature. The results
were compared to the differences in the cuticular patterns of mature and immature individuals.

The reaction of N. vespilloides individuals from the European population towards those of different
species of Nicrophorus was tested to determine whether they were able to distinguish conspecific
potential partners from individuals of other species. The similarity of the cuticular pattern and the
interspecific responses were compared. The method by which individuals may be able to determine
the differences in the cuticular patterns is discussed.

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