Lack of nucleotide variability in a beetle pest with extreme inbreeding.
ABSTRACT The coffee berry borer beetle Hypothenemus hampei (Ferrari) (Curculionidae: Scolytinae) is the major insect pest of coffee and has spread to most of the coffee-growing countries of the world. This beetle also displays an unusual life cycle, with regular sibling mating. This regular inbreeding and the population bottlenecks occurring on colonization of new regions should lead to low levels of genetic diversity. We were therefore interested in determining the level of nucleotide variation in nuclear and mitochondrial genomes of this beetle worldwide. Here we show that two nuclear loci (Resistance to dieldrin and ITS2) are completely invariant, whereas some variability is maintained at a mitochondrial locus (COI), probably corresponding to a higher mutation rate in the mitochondrial genome. Phylogenetic analysis of the mitochondrial data shows only two clades of beetle haplotypes outside of Kenya, the proposed origin of the species. These data confirm that inbreeding greatly reduces nucleotide variation and suggest the recent global spread of only two inbreeding lines of this bark beetle.
- SourceAvailable from: Isabelle Dusfour[Show abstract] [Hide abstract]
ABSTRACT: Anopheles sundaicus s.l. is a malaria vector in coastal areas of Southeast Asia. Previous studies showed at least four distinct species within the complex. The present study investigated the phylogeography and the status of A. sundaicus s.l. populations from Cambodia, Thailand, Malaysia and Indonesia with regard to A. sundaicus s.s. from Sarawak, Malaysian Borneo and A. epiroticus in Vietnam and Thailand. Three lineages recovered by analyses of Cyt-b and COI (mtDNA) confirmed the presence of A. sundaicus s.s. in Malaysian Borneo, the distribution of A. epiroticus from southern Vietnam to peninsular Malaysia, and recognised a distinct form in Indonesia that is named A. sundaicus E. The phylogenetic and demographic analyses suggest that the three species were separated during the Early Pleistocene (1.8-0.78 Myr) and experienced bottlenecks followed by a genetic expansion in more recent times. Based on the results and knowledge of the biogeography of the area, we hypothesise that the combination of cyclical island and refugium creation was the cause of lineage isolation and bottleneck events during the Pleistocene.Infection Genetics and Evolution 08/2007; 7(4):484-93. · 2.77 Impact Factor
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ABSTRACT: Bark beetles are emerging as pests of macadamias, both in the native range of macadamias in Australia and worldwide wherever macadamias are cultivated. Multiple species have been detected on macadamias in Australia; however, little has been known about the identity of the species involved, other than that some belong to the genera Hypothenemus Westwood (1836) and Cryphalus Erichson (1836). Hypothenemus is a large and cosmopolitan genus, which contains two exotic species that are regulated pests for Australia: the tropical nut borer, Hypothenemus obscurus (Fabricius), is a pest of macadamias and Brazil nuts in the Americas and the Pacific, and the coffee berry borer, Hypothenemus hampei (Ferrari), is a pest of coffee found in coffee-growing areas worldwide, but not in Australia. It is essential that biosecurity authorities have reliable species diagnostic tools available in order to detect incursions of these species in Australia. However, the taxonomic literature on the relevant species is scattered and sparse, and the lack of molecular diagnostic methods means that identification of eggs and larvae has been impossible to date because the immature life stages are morphologically homogeneous. This study fills some crucial gaps in our ability to identify these species, developing diagnostic methods for the major pest species on macadamia in Australia, and for key exotic species, including both regulated pests. An integrative taxonomic approach was used incorporating both traditional morphological taxonomy and DNA barcode data in an iterative process to both identify beetles and develop robust diagnostics for them. DNA barcodes provide unambiguous discrimination of all species examined in this study, albeit a limited sample, and have the advantage that they can be used to identify all life stages of the species.Australian Journal of Entomology 04/2010; 49(2):104 - 113. · 0.88 Impact Factor
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ABSTRACT: The resistant Rdl allele for dieldrin insecticide was detected on the Hypothenemus hampei populations from Colombia using conventional PCR methods. Based on this sequence, a melting temperature (Tm) shift genotyping method that relies on allele-specific PCR is described for insecticide resistance-associated single nucleotide polymorphism (SNP) at the H. hampeiRdl gene. The method reported here uses GC-rich tails of unequal length attached to allele-specific primers containing 3′ terminal bases that correspond to SNP allelic variants. Specific PCR products are identified by inspection of a melting curve on a real-time PCR thermocycler using SYBR Green DNA binding dye. Resistant and susceptible alleles resulted in specific PCR products with Tm of 83.3±0.1°C and 86.0±0.2°C, respectively. The RdlTm-shift genotyping method is a new method to identify the Rdl gene in the coffee berry borer H. hampei, the principal pest of coffee that in general show low genetic diversity and very few genetic strategies for control of this pest have been developed. The method supplies a high-throughput tool for dieldrin resistance-associated SNP diagnostic in the coffee berry borer which will be useful for resistance-management strategies and as genetic marker in the colombian insect populations for genetics research.Pesticide Biochemistry and Physiology - PESTIC BIOCHEM PHYSIOL. 01/2010; 97(3):204-208.
Insect Molecular Biology (1998) 7(2), 197-200
back of nucleotide variability in a beetle pest
with extreme inbreeding
\ f i 8
D. Andreev, H. Breilid,* L. Kirkendall: L . O.]Brun't
and R. H. ffrench-Constant
Department of Entomology, University of Wisconsin-
Madison, Madison, Wisconsin, USA, 'Zoology
Museum, University of Bergen, Norway, and
?ORSTOM, Noumea, New Caledonia
The coffee berry borer beetle Hypofhenemus hampei
(Ferrari) (Curculionidae: Scolytinae) is the major
insect pest of coffee and has spread to most of the
coffee-growing countries of the world. This beetle also
displays an unusual life cycle, with regular sibling
mating. This regular inbreeding and the population
bottlenecks occurring on colonization of new regions
should lead to low levels of genetic diversity. We were
therefore interested in determining the level of nucleo-
tide variation in nuclear and mitochondrial genomes of
this beetle worldwide. Here we show that two nuclear
loci (Resisfance to dieldrin and ITS2) are completely
invariant, whereas some variability is maintained at a
mitochondrial locus (COI), probably corresponding to
a higher mutation rate in the mitochondrial genome.
Phylogenetic analysis of the mitochondrial data shows
only two clades of beetle haplotypes outside of Kenya,
the proposed origin of the species. These data confirm
that inbreeding greatly reduces nucleotide variation
and suggest the recent global spread of only two
inbreeding lines of this bark beetle.
Keywords: inbreeding, nucleotide variability, Resis-
tance to die/drin, mitochondrial DNA, coffee berry
borer, Hypothenemus hampei.
The coffee berry borer, Hypothenemus hampei, is the
major insect pest of coffee and has spread to most of
Received 1 1 August 1997; accepted 1 October 1997. Correspondence:
Dr R. H. ffrench-Constant, 237 Russell Laboratories, 1630 Linden Drive,
Madison, WI 53706, USA. e-mail: fi rench@vmsZ.macc.w¡sc.edu.
0 1998 Blackwell Science Ltd
the coffee-growing countries of i..$ world (Le Pelley,
1968). This insect has also recently evolved resistance
to the insecticide endosulfan (a cyclodiene type com-
pound) (Brun et al., 1989, 1990), which is one of the
main chemicals used in control of the borer. As well as
being a remarkably successful insect pest, H, hampeì
also displays an unusual life cycle with regular
inbreeding. Simple population genetic theory suggests
that this regular inbreeding and the founder effects
occurring on the colonization of new regions should
lead to low levels of genetic diversity. We were there-
fore interested in documenting the levels of nucleotide
variability in this inbreeder and in trying to reconcile
the expected reduction in variability with the ecological
success of the insect and its ability to evolve insecti-
The coffee berry borer shows regular inbreeding.
Single mated female H. hampei burrow into coffee
berries and produce large single families with highly
distorted sex ratios (ten females to one male). The
dwarfed males are flightless and mate with their
sisters in the natal berry. The beetle is also 'function-
ally haplodiploid', as paternal genes in males are
neither expressed nor transmitted (Brun et al.,
1995a, b). Thus, both the nuclear and mitochondrial
genome are strictly maternally inherited and should
share an identical genealogy. Resistance to the cyclo-
diene type insecticide endosulfan has also recently
been documented in H . hampei in the South Pacific
island of New Caledonia (Brun et al., 1989, 1990).
Resistance is associated with replacement of a single
amino acid, alanine302 > serine, in the y-aminobutyric
receptor subunit coded for by the ßesisf-
ance to dieldrin gene (ffrench-Constant et al., 1994).
Here we report that both the cyclodiene resistance
gene ßdl itself and a different nuclear locus, the
lntergenic Spacer region (ITS2) of the 5.8s and 28s
ribosomal RNA, show a complete absence of nucleo-
tide variability across the globe (with the exception of
the resistance associated point mutation in ß d l ) : "
Although a mitochondrial locus, cytochrome oxidase 1
(COI) retains some nucleotide variation. Phylogenetic
Fonds Documentaire ORSTOM
D. Andreevet al.
analysis of this mitochondrial DNA variation suggests
that only a few inbreeding strains of the coffee berry
borer have colonized the world. The factors likely to
reduce nucleotide variation in this inbreeder and the
contrast with the remarkable ecological success of this
beetle are discussed.
Results and Discussion
Lack of nuclear nucleotide variation
To test whether or not the level of nucleotide variation
in H, hampei is reduced by inbreeding we examined
two nuclear loci (Rdl and ITS2) and one mitochondrial
locus (COI) via PCR amplification and direct nucleotide
sequencing of the PCR products. We sequenced all
three loci in seventeen strains collected world-wide.
Sequencing of 800 bp from the insecticide resistance
associated locus Resistance to dieldrin (ffrench-
Constant et al., 1994) (Fig. la) revealed no nucleotide
variation, except for the presence or absence of the
insecticide resistance associated nucleotide substitu-
tion. To guard against the possibility that this locus was
under abnormally strong selection (although resistant
strains have only been collected from the South Pacific
island of New Caledonia) we also examined ITS2, a
non-coding region of a second nuclear locus highly
variable in other insects (Fig. 1 b). Sequencing of 747 bp
from ITS2 of all strains also revealed no variation.
Amplification and sequencing of both ßdl and ITS2
from the related inbreeding beetle H. obscurus,
showing 1.9% and 4.0% divergence respectively from
H. hampei, was carried out to guard against unin-
tended repeated PCR amplification of exactly the
zygosity over intial generations (values reducing as
follows for each generation; 1.0, 1.0, 0.75, 0.62, 0.50,
0.40, 0.32, 0.14, 0.04, 0.01 and 0.002) followed by an
average of a 19% loss at each subsequent generation
(Wright, 1921). Such a decline would dramatically
reduce heterozgosity and full-sib mating may therefore
be the dominant factor in reducing nucleotide variation
in H. hampei. (2) Bottlenecks occurring upon coloniza-
tion of new regions. As the spread of H .
countries has been relatively recent, little divergence
within these founder populations would be expected.
This factor would be expected to affect both mitochon-
drial and nuclear genomes. Further, as a result of both
the level of inbreeding and population bottlenecks, as a
párticular mutation fixes in a population, variants
present in the genome in which it arose will also be
fixed, as there is no outcrossing. (3) Finally, the
appearance of deleterious mutations will also elimi-
Figure 1. Diagram showing the relative location and lengths of the
regions sequenced in the two nuclear loci (a) Resistance to dieldrin
(ßdl, DDBLlEMBLlGenBank accession number AF037324) and (b) the
second Intergenic Spacer (ITS2) o f the two ribosomal RNA genes
(accession number AF037326), and (c) !he single mitochondrial locus
cytochrome oxidase I (COI, accession number AF037325). A star
indicates the location o f the resistance associated mutation in Rdl.
same product. We also sequenced 1203 bp from the H.
hampei mitochondrial locus COI (Fig. IC)
twenty-one variable positions (1.8% variation) within
populations. This higher level of variation in the mito-
chondrial genome can be explained by an apparent
rapid rate of evolution (higher mutation rate) in H.
hampei mitochondrial DNA. Thus, the open reading
frame of the mitochondrial COI locus of H. hampei and
H. obscurus shows avery high level of divergence (Ks,
0.84), whereas intron sequence from the nuclear locus
ßdl diverges by only 2%.
Factors contributing to reduction in nucleotide variation
Several factors associated with different insect life cycles
might be expected to reduce genetic polymorphism. In
aphids, for example, these include parthenogenicity
(anholocycly), annual population bottlenecks and founder
effects (Loxdale & Brookes, 1989). Thus in aphids average
biochemical polymorphism (P) is reduced, as is average
heterozygosity (H) with values of approximately 4.5%
against an average o f 7.3% in other insects (Loxdale etal.,
1985). However, two factors relating to these studies are
relevant to the current discussion. Firstly, biochemical poly-
morphism is maintained in aphids even in the presence of
anholocycly. Secondly, many studies of variability in insects
have been restricted to allozyme analysis and the level of
underlying nucleotide variability has not been assessed.
The aim of this study was therefore to quantitate the
reduction in nucleotide variation associated with the
unusual life cycle of the coffee berry borer. Several
factors associated with the life cycle of H. hampei
would be expected to reduce nucleotide variation.
(1) Only single families are usually found in each
coffee berry. Full-sib mating would therefore be
expected to reduce heterozygosity at a rapid rate.
Calculations show a rapid initial decline in hetero-
hampei to many
1998 Blackwell Science Ltd, h e c t Molecular Biology7: 197-200
Nucleotide variability and inbreeding
Thai 1 and
nate the genomes in which they are found via natural
selection, also reducing diversity. Our results on H.
hampei thus confirm the predicted reduction in the
nucleotide variability of this beetle, data also supported
by the apparent lack of variation in a global survey of
sixteen allozymes (Breilid & Kirkendall, unpublished).
Interestingly, despite the apparent absence of
nucleotide variation in the two nuclear loci sequenced,
the resistance associated point mutation in Rdl was
present and has been selected for at least in the South
Pacific island of New Caledonia (ffrench-Constant et
al., 1994). If resistance is absent from the rest of the
world (a systematic survey has not been completed),
this may represent an independent origin of resistance
in a single inbreeding line. However, regardless of the
uniqueness of resistance in New Caledonia, the level
of inbreeding in H. hampei will constrain independent
resistance associated mutations within a limited
number of colonizing lines. In contrast, examination
of a global Rdl phylogeny in an outbreeding flour
beetle, Tribolium castaneum, shows clear evidence
for multiple independent origins of cyclodiene resist-
ance associated mutations on different continents
(Andreev etal., 1997).
Figure 2. Strict consensus tree of COI data
derived by phylogenetic analysis using
parsimony (PAUP version 3.0). Bootstrap
values greater than 50% are given alongside
the number o f nucleotide changes in
parentheses. The tree is rooted to the two
outgroup species Coccotrypes dactyliperda
and Cryphalus af/.
Outside of Kenya (the putative ancestral origin
o f H .
hampei) note the two invariant clades
corresponding to East Africa CentraVSouthern
America and South-East Asia/South Pacific
(including Jamaica and Ivory Coast). Strains
are named according to their country of origin.
The suffixes R I 4 3 and S143 indicate three
cyclodiene resistant and three susceptible
strains collected from New Caledonia.
Global colonization by a few inbreeding lines
Examination of the most parsimonious tree o f the
mitochondrial data (Fig. 2) suggests that outside of
Kenya, the putative ancestral origin of the species, the
world has been colonized by only two inbreeding lines
of H . hampei, one encompassing central and southern
America and the other all strains from south east Asia,
the south Pacific, Jamaica and the Ivory Coast. Two
further factors are of interest in relation to the mito-
chondrial variability. Firstly, on a geographical level,
the particular strain sequenced from Kenya does not
correspond to either of the two colonizing clades. This
may reflect a greater mitochondrial haplotype diversity
in the source country. However, this assumption would
have to be supported by analysis of a larger number
of strains from Kenya. Secondly, given the unusual
chromosome/life cycle o f H. hampei, in which both the
nuclear and mitochondrial genomes are effectively
maternally inherited, it is interesting that variation is
present in the mitochondrial genome but virtually
absent from the nuclear loci examined. This higher
level of mitochondrial variation is probably due to a
higher mutation rate versus the nuclear genome, as
documented for other organisms, or could be main-
H. obscurus 1
0 1998 Blackwell Science Ltd, lnsect Molecular Biology7: 197-200
D. Andreevet al.
tained by independent selection of the two different
mitochondrial haplotypes. The possibility of the mito-
chondria being heteroplasmic (more than one haplo-
type per individual) can be excluded as no hetero-
zygosity was apparent in the sequencing ladders
(direct sequencing of PCR products). Formal estimates
of the expected rate of decline in mitochondrial nucleo-
tide variability are difficult to estimate in the absence of
data relating to the number of mitochondrial particles
transmitted per generation (J. Crow, pers. comm.).
Taken together, these data support a hypothesis of
recent world-wide spread of a few inbreeding coffee
berry borer lines and confirm the predicted theoretical
depression in nucleotide variation associated with
inbreeding and population bottlenecks. Interestingly,
inbreeding has evolved repeatedly in bark beetles: if
other species are similarly genetically depauperate, then
their considerable ecological (Kirkendall, 1993) success
poses an interesting challenge to evolutionary biologists.
Strain collection, PCR amplification and sequencing
H. hampei strains were collected in coffee berries from the
field. Beetles were dissected from the berries in the laboratory
and raised on artificial diet (Brun etal., 1993). Progeny were
bioassayed with endosulfan (Brun et al., 1991) to select for
resistant heterozygotes and strains showing resistance were
then made homozygous by repeated insecticide selection and
crossing of survivors inter se. Genomic DNA was prepared
(Andreev et a/., 1994) from each homozygous strain (strains
consistently surviving a dose of insecticide discriminating
homozygous from heterozygous insects). For PCR, approxi-
mately 100 ng of genomic DNA was added to a 50 p I reaction
containing 0.2 p~ of each primer, 0.2 mM dNTPs and 1.5 units of
Taq polymerase. Samples were denatured at 94°C for 2 min
and then PCR was carried out for 35 cycles of 1 min denatura-
tion at 94"C, 2 min annealing at 50°C and 3 min extension at
72°C. Regions of the three loci were amplified by the PCR and
reaction products sequenced directly withoutcloning (to avoid
polymerase errors often associated with individual cloned
products). PCR primers used for ßdl were: forward
forward GTGGATCCTGTGAACTGCAGGACACATG, reverse
GTGAATTCATGCTTAAATTTAGGGGGTA and for COI: forward
GGATCACCTGATATAGCATTCCC, reverse GTTTAAGAGAC-
CAGTACTTG. PCR products were sequenced using an ABI
373 automated sequencer (Applied Biosystems).
Phylogenetic analysis of COI data
A strict consensus tree o f COI data was derived by phylo-
genetic analysis using parsimony using the PAUP software
package version 3.0. Bootstrap values were calculated and
those greater than 50% are given alongside the number of
nucleotide changes in parentheses on the tree (Fig. 2).The tree
is rooted to the two outgroup beetle species Coccotrypes
dactyliperda (F.) and Cryphalus aft indicus (Eichoff).
We thank J. Crow, M. Kreitman, B. Charlesworth and
D. Charlesworth for help with data analysis and dis-
cussion of the data and O. Andreev for assistance with
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1998 Blackwell Science Ltd, lnsect Molecular Biology7: 197-200
Volume7 0 Number2
CODEN IMBIE3 ISSN 0962-1075
Editors: J. M. Crampton and A. A. James
Published for the Royal Entomological Society