The principal malaria vector in Africa, Anopheles gambiae, contains two pairs of autosomes and one pair of sex chromosomes. The Y chromosome is only associated with males and other Y chromosome-specific DNA sequences, which are transferred to women during mating. A reliable tool to determine the mating status of dried wild An. gambiae females is currently lacking. DNA was extracted from dried virgin and mated females and used to test whether Y chromosome-specific polymerase chain reaction (PCR) markers can be successfully amplified and used as a predictor of mating. Here we report a new PCR-based method to determine the mating status among successfully inseminated and virgin wild An. gambiae females, using three male-specific primers. This dissection-free method has the potential to facilitate studies of both population demographics and gene flow from dried mosquito samples routinely collected in epidemiologic monitoring and aid existing and new malaria-vector control approaches

Horton, Ashley

Knols, Bart G J

Lanzaro, Gregory C

Ng'habi, Kija R

English

The principal malaria vector in Africa, Anopheles gambiae, contains two pairs of autosomes and one pair of sex chromosomes. The Y chromosome is only associated with males and other Y chromosome¿specific DNA sequences, which are transferred to women during mating. A reliable tool to determine the mating status of dried wild An. gambiae females is currently lacking. DNA was extracted from dried virgin and mated females and used to test whether Y chromosome¿specific polymerase chain reaction (PCR) markers can be successfully amplified and used as a predictor of mating. Here we report a new PCR-based method to determine the mating status among successfully inseminated and virgin wild An. gambiae females, using three male-specific primers. This dissection-free method has the potential to facilitate studies of both population demographics and gene flow from dried mosquito samples routinely collected in epidemiologic monitoring and aid existing and new malaria-vector control approaches

Ng'habi, K.R.

Horton, A.

Knols, B.G.J.

Lanzaro, G.C.

NARCIS 

A new robust diagnostic polymerase chain reaction for determining the mating status of female Anopheles gambiae mosquitoes

Digital Library of the Tanzania Health Community

A New Robust Diagnostic Polymerase Chain Reaction for Determining the Mating
Status of Female Anopheles gambiae Mosquitoes
Kija R. Ng’habi,* Ashley Horton, Bart G. J. Knols, and Gregory C. Lanzaro
Ifakara Health Research and Development Centre, Tanzania; Center for Vectorborne Diseases, University of California, Davis,
California; Wageningen University and Research Centre, Wageningen, The Netherlands
Abstract. The principal malaria vector in Africa, Anopheles gambiae, contains two pairs of autosomes and one pair
of sex chromosomes. The Y chromosome is only associated with males and other Y chromosome–specific DNA
sequences, which are transferred to women during mating. A reliable tool to determine the mating status of dried wild
An. gambiae females is currently lacking. DNA was extracted from dried virgin and mated females and used to test
whether Y chromosome–specific polymerase chain reaction (PCR) markers can be successfully amplified and used as a
predictor of mating. Here we report a new PCR-based method to determine the mating status among successfully
inseminated and virgin wild An. gambiae females, using three male-specific primers. This dissection-free method has the
potential to facilitate studies of both population demographics and gene flow from dried mosquito samples routinely
collected in epidemiologic monitoring and aid existing and new malaria-vector control approaches.
INTRODUCTION
Current knowledge suggests that sex chromosomes of many
groups of animals and plants evolved from a pair of ordinary
autosomes that acquired a major sex-determining locus.
1,2
The Y chromosome, frequently associated with sex determi-
nation, and its associated genes have been well explored in a
range of vertebrate organisms.
3 Natural selection is believed
to have suppressed the ability of the Y chromosome to re-
combine with its counterpart.
4 Recombination suppression is
considered to be responsible for a gradual Y chromosome
genetic degradation.
5 The silencing of most of the genes
present on the Y chromosome
6,7 as a result of degradation
explains why only a few male fertility–determining genes re-
main functional on this chromosome.
8,9 Among invertebrates,
Y-linked gene studies have been limited to Drosophila
10 and
were recently extended to An. gambiae.
11,12
The An. gambiae mosquito has a karyotype consisting of
two pairs of autosomes chromosomes 2 and 3 and one pair of
sex chromosomes chromosomes X and Y. The Y chromo-
some constitutes ∼10% of the whole genome and is normally
associated only with males, and when present in a XX/XY
system, induces male development.
13 Y chromosome–linked
DNA fragments have been characterized and Y chromo-
some–specific polymerase chain reaction (PCR) markers
have been developed.
11,12 This finding has been viewed as a
milestone toward the application of novel vector control strat-
egies for which a thorough understanding of population struc-
ture and gene flow among An. gambiae populations is neces-
sary.
Traditionally, detection of mating success among females
relied on microscopic dissection of females to determine the
presence or absence of a mating plug in the bursa copulatrix
or examination of sperm in the female spermatheca. This
method is reliable and robust but is time consuming, labor
intensive, and requires fresh specimens. A simple and rapid
method to determine the mating status of dried female An.
gambiae is therefore required to analyze large sample sizes
within a short period of time.
In this study, we tested the hypothesis that Y chromosome–
specific PCR markers can be successfully amplified from male
sperm DNA present in recently inseminated females of An.
gambiae sensu stricto and An. arabiensis. We present this
technique as a new dissection-free (PCR based) method to
determine the mating status of samples collected during rou-
tine entomologic surveys.
MATERIALS AND METHODS
Experimental design. Mosquitoes used in this study were
from laboratory colonies obtained from the Malaria Research
and Reference Reagent Resource Center (MR4) and main-
tained at the University of California, Davis, CA. These in-
cluded a colony of Anopheles gambiae s.s. originally collected
from Kisumu (East Africa) and a colony of An. arabiensis
(Dongola strain) originating from northern Sudan. Pupae of
both An. gambiae s.s. and An. arabiensis were isolated in
cages allowing males and females to emerge separately. In
this experiment, 100 female pupae and 50 male pupae were
isolated to provide an appreciable number of virgin females
and males, respectively. After adult emergence, 25 virgin fe-
males were mixed with 25 males for 3 days and exposed to
natural sunlight (dusk) to stimulate mating. Another 25 virgin
females were kept in a separate cage for 3 days as a control.
On Day 3, 15 virgin females, 15 potentially mated females,
and 10 males were killed and stored at 4°C. After 5 days,
when the samples had dried, DNA was extracted from the
whole body of males and virgin and potentially mated fe-
males, following the DNAzol extraction protocol.
14 A sub-
sample of 10 potentially mated females were dissected under
the microscope to confirm insemination by examination of
the spermatherca. The experiment was done in triplicate.
The PCR reactions consisted of the following concentra-
tions. 5 L of 10× reaction buffer containing 1.5 mmol/L of
MgCl2, 0.2 L of each DNPT at a concentration of 2.5 mmol/
L, 25 pmol of each primer, and 0.5 Lo fTaq polymerase in
a total of 50 L. Primer sequences to amplify DNA have been
published for An. gambiae.
11 Primer pairs were S23 (635 bp;
F: 5-CAAAACGACAGCAGTTCC-3;R :5 -TAAACCAA-
GTCCGTCGCT-3), 128125B (547 bp; F: 5-AGCGTGGAG-
GACATACAA-3;R :5 -ATGGCAATTTCGTTTTCA-3),
and 128125I (581 bp; F: 5-GGCCTTAACTAGTCGGGTAT-
3;R :5 -TGCTTTCCATGGTAGTTTTT-3) as described.
11
* Address correspondence to Kija R. Ng’habi, Ifakara Health Re-
search and Development Centre, PO Box 53, Ifakara, United Repub-
lic of Tanzania. E-mail: kija@ihrdc.or.tz
Am. J. Trop. Med. Hyg., 77(3), 2007, pp. 485–487
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene
485Thermal cycling for all reactions were performed using an MJ
Research PTC-225 tetrad thermocycler. Conditions were the
same for all primers (S23, 128125B, and 128125I): 94°C for 3
minutes, 35 cycles of 94°C for 20 seconds, 55–64°C for 30 sec-
onds, and 72°C for 1 minute, followed by 72°C for 10 minutes.
Loading buffer (6×) and 3% agarose gel was used. The agarose
gel was submerged in the electrophoresis chambers, two thirds
filled with 10× TBE running buffer. The gel was run for 1.5
hours at 85 mA.
RESULTS
We performed experiments to determine whether a multi-
copy locus (ribosomal DNA) can be amplified in inseminated
females. The experiment determined whether the DNA from
the male partner of mated An. gambiae s.s. and An. arabiensis
females could be amplified by PCR. A total of 50 virgin fe-
males, 57 potentially mated females, and 28 males of both An.
gambiae and An. arabiensis were used in the study. Primers
S23 and 128125B gave strong amplification in An. gambiae s.s.
females, whereas primer 128125I gave the strongest amplifi-
cation in An. arabiensis females. DNA from dried specimens
amplified in a similar way as those from fresh specimens. All
dissected females were found to have sperm in their sper-
mathecae. Figure 1, A and B, shows that male DNA from
inseminated females of both species can be amplified using Y
chromosome–specific PCR markers with similar efficiency as
the males themselves. As expected, no amplification was ob-
tained from virgin females.
DISCUSSION
We showed that this method is suitable for distinguishing
mated females from virgin females for both An. gambiae s.s.
and An. arabiensis specimens and that this approach lends
itself to screening large numbers of recently mated and dried
specimens. This finding has useful implications for novel vec-
tor control strategies based on the release of large numbers of
factory-reared male mosquitoes with the expectation that
they will successfully mate with wild females to confer sterility
or drive parasite-refractory genes into wild populations. In
these cases, the transfer of sterile or transgenic sperm from
released mosquitoes to wild conspecifics will depend on the
frequency of mating between males carrying the trait (to be
introduced) and the females that do not—even if target genes
are linked to transposable elements.
15 Because mating is ul-
timately the most appropriate mechanism to deliver the de-
sired traits or genes into a target population,
16 an effective
method for evaluating mating success is of critical importance.
Thus, this new dissection-free technique has potential useful
application to novel vector control approaches.
Additionally, this technique will help improve our under-
standing of gene flow within An. gambiae populations. As
opposed to methods based on microscopic dissection and di-
rect observation that are conventionally used to assess male
An. gambiae mating success, this method will make it possible
to screen a substantially greater number of wild-collected fe-
males either in fresh or dried state. Contained semi-field sys-
tems are now becoming a central focus for release trials in
many studies.
17–19 The method developed here can thus make
large-scale evaluation of mating success of released males by
repetitive female sampling and screening possible.
In conclusion, we showed that mating can be determined by
PCR amplification of inseminated female DNA. The speed
and ease of this technique, and the fact that dried specimens
can easily be evaluated, indicate that this process should allow
robust and extensive analysis of large field-collected samples.
Received January 31, 2007. Accepted for publication April 30, 2007.
Acknowledgments: We thank the Vector Genetic Laboratory (Uni-
versity of California) for cooperation during the entire period of this
study; Claudio Meneses for invaluable technical assistance during
mosquito rearing and PCR optimization; Dr. Mark Benedict for sug-
gestions and literature that made this study possible; and Dr. Heather
Ferguson for reviewing the manuscript before submission.
Financial support: We acknowledge the International Atomic Energy
Agency (IAEA) for financial support through a fellowship awarded
to KRN to work at UC Davis. This research forms part of a VIDI
Grant 864.03.004 from the Dutch Scientific Organization (NWO)
awarded to BGJK. In addition, we acknowledge support to GCL
through Grant AI040308 from the National Institutes of Health.
Authors’ addresses: Kija R Ng’habi, Ifakara Health Research and
Development Centre, PO Box 53, Ifakara, United Republic of Tan-
zania, E-mail: kija@ihrdc.or.tz. Ashley Horton and Gregory Lanzaro,
Department of Entomology, University of California, Davis, 396C
Briggs, One Shield Avenue, Davis, CA 95616-8584, E-mails:
gclanzaro@ucdavis.edu and ahorton@ucdavis.edu. Bart G. J. Knols,
Laboratory of Entomology. PO Box 8031, 6700 EH, Wageningen
University and Research Centre, Wageningen, The Netherlands, E-
mail: Bart.Knols@wur.nl.
REFERENCES
1. Muller HJ, 1932. Some genetic aspects of sex. Am Nat 66: 118–
138.
2. Charlesworth B, Charlesworth D, 2000. The degeneration of Y
chromosomes. Philos Trans R Soc Lond B Biol Sci 355: 1563–
1572.
3. White MJD, 1973. Animal Cytology and Evolution. Cambridge:
University Press.
4. Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum HS, Hillier
L, 2003. The male-specific region of the human Y chromosome
is a mosaic of discrete sequencie classes. Nature 423: 825–837.
5. Bachtrog D, 2003. Accumulation of spock and worf, two novel
non-LTR retrotransposons on the neo Y-chromosome of
Drosophila miranda. Mol Biol Evol 20: 173–181.
6. Bachtrog D, 2003. Adaptation shapes pattern of genome evolu-
FIGURE 1. Agarose gel electrophoresis showing amplification of
Y chromosome sequences in (A) An. arabiensis (primer 128125I) and
(B) An. gambiae s.s. (Primer S23) males and mated females. There
was no amplification in virgin females.
NG’HABI AND OTHERS 486tion on sexual and asexual chromosomes in Drosophila. Nat
Genet 34: 215–219.
7. Steinemann S, Steinemann M, 2000. Y chromosomes: born to be
destroyed. Bioessays 27: 1076–1083.
8. Carvalho AB, Lazzaro BP, Clack AG, 2000. Y-chromosomal fer-
tility factors kl-2 and kl-3 of Drosophila melanogaster encode
dynein havy chain polypeptides. Proc Natl Acad Sci USA 98:
13225–13230.
9. Carvalho AB, Dobo BA, Vibranovski MD, Clack AG, 2001.
Identification of five new genes on the Y-chromosome of
Drosophila melanogaster genome: how far can we go? Genetica
117: 227–237.
10. Steinemann M, Steinemann S, 1998. Enigma of Y-chromosome
degeneration: neo-Y and neo-X chromosomes of Drosophila
miranda a model for sex chromosome evolution. Genetica 102/
103: 409–420.
11. Krzywinski J, Nusskern DR, Kern MK, Besansky NJ, 2004. Iso-
lation and Characterization of Y chromosome sequences from
the African malaria mosquito Anopheles gambiae. Genetics
166: 1291–1302.
12. Krzywinski J, Sangare D, Besansky NJ, 2005. Satellite DNA from
the Y-chromosome of the malaria vector Anopheles gambiae.
Genetics 169: 185–196.
13. Clements AN, 1992. The Biology of Mosquitoes, Vol 1: Develop-
ment, Nutrition and Reproduction. London: Chapmann and
Hall.
14. Chomczynski P, Mackey K, Drews R, Wilfinger W, 1997. DNA-
zol: a reagent for the rapid isolation of genomic DNA. Bio-
techniques 22: 550–553.
15. Tabachnick WJ, 2003. Reflections on the Anopheles gambiae ge-
nome sequence, transgenic mosquitoes and the prospect for
controlling malaria and other vector borne diseases. J Med
Entomol 40: 597–606.
16. Catteruccia F, Godfray HCJ, Crisanti A, 2003. Impact of genetic
manipulation on the fitness of Anopheles stephensi mosquitoes.
Science 299: 1225–1227.
17. Knols BGJ, Njiru BNN, Mathenge EM, Mukabana WR, Beier
JC, Killeen GF, 2002. Malariasphere: a greenhouse-enclosed
simulation of a natural Anopheles gambiae (Diptera: Culi-
cidae) ecosystem in Western Kenya. Malar J 1: 19.
18. Ferguson FM, John B, Ng’habi KR, Knols BGJ, 2005. Addressing
the sex imbalance in knowledge of vector biology. Trends Ecol
Evol 20: 202–209.
19. Knols BGJ, Njiru BNN, Mukabana RW, Mathenge EM, Killeen
GF, 2003. Contained semi-field environments for ecological
studies on transgenic African malaria vectors. Scott TW,
Takken W, eds. Ecology of Transgenic Mosquitoes. Wagenin-
gen: Wageningen University and Research Centre, 99–106.
PCR FOR MATING DETECTION OF AN. GAMBIAE 487

Accumulation of spock and worf, two novel non-LTR retrotransposons on the neo Y-chromosome of Drosophila miranda.

Addressing the sex imbalance in knowledge of vector biology.

Animal Cytology and Evolution.

Contained semi-field environments for ecological studies on transgenic African malaria vectors.

DNAzol: a reagent for the rapid isolation of genomic DNA.

Enigma of Y-chromosome degeneration: neo-Y and neo-X chromosomes of Drosophila miranda a model for sex chromosome evolution.

Identification of five new genes on the Y-chromosome of Drosophila melanogaster genome: how far can we go?

Impact of genetic manipulation on the fitness of Anopheles stephensi mosquitoes.

Isolation and Characterization of Y chromosome sequences from the African malaria mosquito Anopheles gambiae.

Malariasphere: a greenhouse-enclosed simulation of a natural Anopheles gambiae (Diptera: Culicidae) ecosystem in Western Kenya.

Reflections on the Anopheles gambiae genome sequence, transgenic mosquitoes and the prospect for controlling malaria and other vector borne diseases.

Satellite DNA from the Y-chromosome of the malaria vector Anopheles gambiae.

Some genetic aspects of sex.

The Biology of Mosquitoes, Vol 1: Development, Nutrition and Reproduction. London: Chapmann and Hall.

The degeneration of Y chromosomes.

The male-specific region of the human Y chromosome is a mosaic of discrete sequencie classes.

Y chromosomes: born to be destroyed.

Y-chromosomal fertility factors kl-2 and kl-3 of Drosophila melanogaster encode dynein havy chain polypeptides.

A new robust diagnostic polymerase chain reaction for determining the mating status of female Anopheles gambiae mosquitoes.

A new robust diagnostic polymerase chain reaction for determining the mating status of female Anopheles gambiae mosquitoes.

Abstract

Similar works

Full text

Available Versions

NARCIS

Digital Library of the Tanzania Health Community