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First published online March 16, 2007
Journal of Experimental Biology 210, 1266-1274 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.002642
Stable isotope-mass spectrometric determination of semen transfer in malaria mosquitoes
1 International Atomic Energy Agency (IAEA), Agency's Laboratories
Seibersdorf, A-2444 Seibersdorf, Austria
2 Laboratory of Entomology, Wageningen University and Research Center, PO
Box 8031, 6700 EH Wageningen, The Netherlands
* Author for correspondence (e-mail: bart.knols{at}wur.nl)
Accepted 5 February 2007
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13C values. Labelling during
larval development and combined labelling of larvae and adults resulted in
detectable values. The label persisted in spermathecae for up to 7 days after
mating, and unlabelled sugar feeding of males labelled in the larval stage did
not result in a detectable turnover of the semen label. There were no
detrimental effects of the addition of labelled glucose on larval development
and survival, adult size, male longevity and mating performance. We have
proved that it is possible to label male mosquitoes and detect the semen label
in females after insemination. This method offers great potential to study
mating in mosquitoes and other insects and could prove useful in genetic
control studies of medical or agricultural pest insects, with male mating
success in the field as a critical verifiable indicator for a positive outcome
of the intervention.
Key words: stable isotope, semen-label, anopheles, mass spectrometry, genetic control
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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).
In insect studies, stable isotopes have not been used extensively, but they
can be a useful tool to address issues of food preference, resource
allocation, dispersal, etc. (Hood-Nowotny
and Knols, in press). Recent studies undertaken in our laboratory
have documented the successful application of stable isotopes as a population
marker in the context of genetic control studies [e.g. Sterile Insect
Technique (Hood-Nowotny et al.,
2006)]. Our current interest lies in the use of stable isotopes to
study mating behaviour. Studies in which natural abundance levels of stable
isotopes were used in the context of mating have been carried out
(Ponsard et al., 2004;
Malausa et al., 2005), but to
our knowledge no work has been performed on the enrichment of insect semen
with stable isotopes. Semen labelling has recently been performed in humans;
2H2O was ingested daily and spermatogenesis kinetics
studied (Misell et al.,
2006).
In the present study we used the African malaria mosquito Anopheles
arabiensis Patton. Of the major life history behaviours of anopheline
mosquitoes, mating remains the least understood
(Takken and Knols, 1999).
Studies on mosquito mating behaviour are difficult to conduct because of its
crepuscular nature, complex constituency (i.e. swarm makeup), and irregular
spatial occurrence. In the context of genetic control studies, understanding a
male's mating success in the field is critical for a positive outcome of the
intervention (Ferguson et al.,
2005), and techniques that label semen (i.e. spermatozoa and
accessory gland fluid) would greatly facilitate the study of mating behaviour.
Radioactive isotopes have been used in the past to study the fate of semen
(Dame and Schmidt, 1964;
Tantawy et al., 1967;
Smittle et al., 1969;
Young and Downe, 1978) in
which mosquitoes were labelled by exposing larvae to radioactive solutions.
This resulted in the transfer of radioactive semen during copulation and the
successful identification thereof in spermathecae. Nowadays, the use of
radioactive isotopes is rarely practised in entomological research due to the
hazards related to the treatment process and environmental concerns of
releasing such insects, even though the half-life of the commonly used isotope
32P is short (e.g. 14.3 days). Other options to study mating in
mosquitoes include the use of mutant strains
(Mason, 1967;
Gomulski, 1988;
Beard et al., 1995;
Klowden, 2006), but these are
not readily available for most stocks. A transgenic strain that can be used to
study mating behaviour has been developed in A. stephensi
(Catteruccia et al., 2005), but
this technology hinges on ethical, legal and social issues affecting the
ability to release transgenic insects
(Knols and Louis, 2006;
Knols et al., 2006), and is
not easily transferable to other species.
Stable isotopes are naturally occurring in the environment, are not
radioactive and therefore do not decay. In general, they react chemically in a
manner identical to the more common isotope and thus are effective,
non-invasive markers in biological systems. Besides these attributes, stable
isotopes are not species-specific, which makes them attractive for use. Most
elements of biological interest (including C, H, O, N and S) have two or more
stable isotopes, with the lightest of these present in much greater abundance
than the others. An isotope of an element has the same atomic number but a
different number of neutrons and consequently a different atomic mass. If a
system is enriched with the less abundant isotope, this element can be used as
a label or tracer (Hood-Nowotny and Knols,
in press). The isotopic composition of a sample is measured by
determining the ratios of the stable isotope masses. These ratios are measured
on an isotope ratio mass spectrometer, a device that separates ions of the
element of interest on the basis of their differing mass/charge ratio
(m/z) (de Groot,
2004).
For the experiments presented in this paper 13C was chosen as a label. The findings of three sets of experiments that address four objectives are presented. The first objective was to see whether it was possible to use 13C as a semen-labelling technique and to identify the optimal developmental stage for labelling. The second objective was to test the ability of males of different ages to transfer the label. The third objective was to test the persistence of the label in spermathecae after mating. The fourth objective was to study the impact of the stable isotope on the mosquito to assure that no detrimental effects occurred.
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Mosquitoes
The Dongola strain of Anopheles arabiensis Patton was used. It was
collected in Northern State, Sudan, in 2004 and has been reared in our
laboratory since then. Five hundred L1 stage larvae were counted and placed in
a tray (30x40 cm) in 1 l of deionized water, and the water level was
kept constant throughout the experiment. Heating mats were used to maintain
the water temperature at 28±1°C. Larvae were fed a diet of fish
food (AquariCare Koi Floating Blend, USA) daily (0.25 mg per larva), that was
ground and sieved through a 224 µm sieve, and mortality of the larvae was
not taken into account. Adults were kept in standard 30 cmx30
cmx30 cm mosquito rearing cages and maintained at a temperature of
27±0.2°C and relative humidity of 80±2%. The light regime
was 10 h:12 h L:D with a 1 h simulated dusk and dawn period. Adults were
maintained on a standard 10% sucrose solution (w/v) unless stated
otherwise.
Labelling
99 atom% [13C]glucose (U-13C6; Cambridge Isotope Laboratories
Inc., Andover, MA, USA) was used as a label. Mosquitoes were exposed to the
label either as larvae or adults. In the larval stage, the label was added to
the water on the same day as the L1 larvae were introduced. In the adult
stage, the label was added to the sugar water. The level of enrichment in both
treatments was 20 atom% 13C (i.e. approximately 20% of all the
carbon in the diet was 13C), and was based on findings from a
previous study (Hood-Nowotny et al.,
2006) where lower levels of enrichment were used (e.g.
1.11–1.46 atom% 13C) for whole body analysis.
The amount of [13C]glucose needed was based on the total amount of carbon present in the diet. 40% of the larval diet consisted of carbon. Until pupation, 1.0 g (0.25 mgx500 larvaex8 days) of larval food was added to the tray, thus 0.1 g of 13C was required. As the percentage of carbon in glucose is 40%, 0.25 g of 99 atom% [13C]glucose was added to the larval trays. For adult mosquito labelling, the stable isotope was incorporated in the sugar solution, and similar calculations were made to determine the amount of label needed. 1.00 g of sucrose has 0.42 g of C, thus 0.1 g of 13C was required. Labelled sugar feeders received 0.25 g of 99 atom% [13C]glucose+1.00 g sucrose in 12 ml water (10.4% sugar solution); and the unlabelled feeders received unlabelled glucose instead.
Experimental design
Males that emerged from the experimental larval trays [e.g. labelled (L)
and unlabelled (U)] were divided into four adult treatments: U–U, males
unlabelled in the larval stage fed on unlabelled sugar; U–L, males
unlabelled in the larval stage fed on labelled sugar; L–U, males
labelled in the larval stage fed on unlabelled sugar; L–L, males
labelled in the larval stage fed on labelled sugar.
When treatments were compared within an experiment, only males emerging on the same day from labelled and unlabelled trays were used. Males were transferred from the larval trays to adult cages and fed on their designated sugar source (e.g. labelled or unlabelled) until mating was initiated. In the experiments where the adult sugar water was labelled, the unlabelled treatments received unlabelled glucose; however, when adult labelling was not performed, cages were maintained on the laboratory standard (e.g. unlabelled) sucrose solution.
When adult labelling was performed, males were transferred to a new cage prior to mating to prevent cross-contamination of the females (e.g. spills from the sugar source), and males in other treatments tested at the same time were also transferred for comparison sake. During and after mating, males were maintained on standard sucrose solution. Females used as mates were isolated within 18 h after emergence to assure virginity. The age of the females in all experiments was similar to that of males when mating was initiated. After mating, females were either: dissected immediately (e.g. the following day) at the end of the mating period (I); or isolated for dissection at a later stage to assess the persistence of the label in the spermathecae (II).
Experiment 1
The main goal of the first experiment was to see if 13C could be
used as a semen label, and to determine the optimal treatments to deliver
[13C]glucose. In addition, persistence of the label in spermathecae
was studied.
For each larval treatment (e.g. labelled glucose and unlabelled glucose) two trays were set up. Males emerging from the trays were pooled according to treatment and divided into the four adult treatments as described above. Males were fed sugar from their designated source for 4 nights. On the fifth day, mating was initiated. Per treatment, 62 males were mated with females at a ratio of 2:1 (M:F) and the mating period lasted for 3 nights. After mating, females were immediately dissected for analyses, or isolated and dissected 3 days later.
A small number of experimental males were removed from the cages on day 4 (L–U, L–L treatments) and day 11 (all treatments) after emergence and their reproductive system dissected for isotopic determination.
Experiment 2
In the second experiment, lower insemination of the females was pursued to
determine if inseminated spermathecae could successfully be distinguished from
uninseminated spermathecae within the same treatment. Treatments from
experiment 1 (U–U, L–U, L–L) were repeated with the
exception of adult only labelling (U–L), and the persistence of the
label in the females and the persistence of the label in males that mated
later in life was investigated. In addition, the impact of labelling on larval
development and adult longevity was studied.
Larval trays included one tray with labelled glucose, and one with unlabelled glucose. Another tray without any glucose was added to monitor the effect of glucose on larval survival. Adults emerging from the trays were removed and counted daily, and trays were maintained until all larvae had pupated and emerged, or died.
The males emerging from the labelled glucose tray were either fed unlabelled or labelled sugar, males from the unlabelled tray were fed unlabelled sugar. After 5 days of sugar feeding, 35 males were mated for 1 night with females on a 1:1 ratio. Females were dissected immediately or isolated and dissected 3 days later (Fig. 2A). Mortality of the males was scored regularly until the majority had died.
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A third batch of males that emerged from the labelled tray was used to study the effect of male age on label transfer. Males were maintained on standard 10% sucrose solution and mated (N=25) with females at a ratio of 1:2 (M:F) on day 4 for 3 nights, or on day 10 (N= 25) for 1 night at a ratio of 1:1. Females were dissected immediately or after 4–5 days of isolation (Fig. 2C).
Experiment 3
The impact of labelling on larval development and adult longevity was
investigated, to complement initial data gathered in experiment 2. In
addition, the effect of 13C on the size of the emerged adults, and
the impact of the semen label on the hatchability of eggs was studied.
Larval treatments included trays with [13C]-labelled glucose, unlabelled glucose, and a control tray; each treatment was duplicated. Adults emerging from the trays were removed and counted daily, and trays were maintained until all larvae had pupated and emerged, or died. On the day the majority of pupae emerged, 50 males were collected per treatment and per replicate and placed in a standard cage to monitor survival. Males were maintained on a standard 10% sucrose solution, and mortality was scored regularly until all males had died.
Adult body size of males and females emerging from the trays was determined
by wing length (Lyimo and Takken,
1993; Lounibos et al.,
1995; Charlwood et al.,
2002). Day of emergence was noted and for each specimen a wing was
clipped and mounted on a slide. A digital image of the wing was taken [CC-12
camera (Olympus Soft Imaging Solutions, Berlin, Germany) mounted on a stereo
microscope]. Wing length was measured between the alula notch and the wing
tip, excluding scales; measurements were performed with AnalySIS FIVE software
(Olympus Soft Imaging Solutions).
The effect of the 13C label on sperm viability was monitored by assessing the hatching of eggs fertilized by labelled sperm. Unlabelled virgin females (N=50) were mated to males emerging from labelled or control trays at a 1:1 ratio and cages were maintained for 26 days. Mosquitoes were membrane blood fed on multiple occasions and eggs were collected en masse and checked for hatching.
Sample preparation
Females
Females were immobilized, and their spermathecae dissected in mosquito
saline (Ephrussi and Beadle,
1936). Insemination status was checked under a compound microscope
at 100x magnification and recorded. The spermatheca was then transferred
to a small piece of quartz fibre filter paper with a fine brush and placed in
a cylindrical 8x5 mm (height x diameter) tin cup. After each
dissection, tools were cleaned with ethanol to avoid contamination. The amount
of carbon present in the spermatheca (
3 µg) was below the detection
limit of the mass spectrometer setup (approx. >20 µg). Samples were
therefore `spiked' with 10 µl of a standard sucrose solution containing
23–26 µg of carbon (Dube et
al., 1998). All samples were dried for 
24 h in an oven at
50°C before closure of the tin cup and subsequent analyses in the mass
spectrometer. Standards containing only the piece of quartz fibre filter paper
with the spike were included. Spermathecae from virgin females were used as a
control and dissected similarly to those of experimental females.
Males
A number of males from the first experiment were dissected to analyse the
amount of 13C in their reproductive system. The testes, accessory
glands and seminal vesicle were dissected and prepared for analyses as above
(e.g. including the spike).
Sample analyses and interpretation
After drying, tin cups were sealed and contents analysed using a Carlo Erba
(Milan, Italy) carbon nitrogen (CN) analyser, linked to an Optima, (Micromass,
Manchester, UK) isotope ratio mass spectrometer (IRMS); see
(Hood-Nowotny et al., 2006)
for details.
The output from the mass spectrometer is a 13C value,
which represents the ratio of 13C over 12C against a
reference standard, and the total amount of carbon present in the sample. The
actual delta value of the sample alone, e.g. without the spike, was not
determined for the spermathecae samples because of uncertainty associated with
calculating this value. This was due to the unknown and low amount of carbon
in the original unspiked sample and proximity of unlabelled treatment
13C values to the 13C of the spike, which
made comparison of labelled and unlabelled samples difficult. Therefore,
spiked 13C values were used for statistical analysis and
data representation. For the males, the actual 13C values
were calculated to obtain an estimate of the enrichment.
Data analyses
Prior to analyses, data were checked for normality. When homogeneity of
variances was not assumed, and the number of treatments exceeded two,
non-parametric tests were performed. Data on spermathecae labelling were
analysed using the following variables: insemination status (inseminated or
uninseminated as determined by compound microscopy) adult labelling treatment
(U–U, U–L, L–U, L–L), and dissection history (I or
II). Some outliers in 13C values were observed in the
dataset, in particular in the first experiment, and these were excluded to
normalise the data (see Results section). Differences between mean
13C values of labelling treatments were analysed using
general linear models (GLMs) with planned contrasts (Tukey's HSD) and data on
dissection history were analysed with GLMs or independent t-tests.
Independent t-tests were also used to compare uninseminated females
to virgin control samples in all experiments, and to test the difference in
13C values of inseminated and uninseminated spermathecae for
each labelling treatment in experiment 2. A threshold value to distinguish
labelled spermathecae from unlabelled spermathecae was defined as 2 or 3
standard deviations (s.d.) above the mean 13C (
)
value of the reference standard (Macneale
et al., 2005), in our case virgin females. Longevity of males was
analysed using Kaplan-Meier survival analyses. The obtained survival curves
were pairwise compared using Mantel-Cox log-rank tests. Carbon data, larval
survival and wing length data were analysed with GLMs. All two-sided tests
were performed using the SPSS software version 12 (SPSS Inc., Chicago,
USA).
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13C values reported are negative as they are
referenced to an international standard PDB (Pee Dee Belemite), which is more
enriched in 13C than our spiked samples.
Optimal treatment
When females from experiment 1 were dissected immediately after mating (I),
sufficient amounts of labelled semen were transferred in all labelled
treatments to distinguish mean 13C values of inseminated
spermathecae from unlabelled samples (F3,38=26.88;
P<0.01) (Fig. 1).
Labelling in the larval stage (L–U) or in both stages (L–L)
resulted in the highest amount of label transferred, but labelling of only the
adult stage (U–L) was sufficient to distinguish mean
13C values from the control (U–U). However, the
persistence of the label in males and females after adult labelling alone was
not sufficient (see below). Therefore, labelling in the adult stage alone was
not considered optimal and further experiments focused on the males labelled
as larvae or as larvae and adults. The second experiment repeated the
treatments of the first experiment, except for the adult only labelling, with
similar results. Mean 13C values of spermathecae inseminated
by males labelled in the larval or in both stages were higher than the control
males after immediate dissection (I) (F2,16.08=160.53,
P<0.01), and in this experiment males labelled in both stages
transferred significantly more label than larval labelled males alone
(Fig. 2A).
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Persistence of label in spermathecae
After isolation of the females for 3 nights (II), mean
13C values decreased in all labelled treatments compared to
immediate dissection (I) in experiment 1
(Fig. 1), and in the treatments
U–L and L–U a significant decrease was observed; U–L:
[t(16)=4.19, P<0.01], L–U: [t(13)=2.43,
P<0.05; Fig. 1].
When males were only labelled in the adult stage, 13C values
of females isolated for 3 days were still higher, but no longer statistically
different from the control samples (F3,22=27.22,
P<0.01), whereas in the larva-labelled treatments mean
13C values remained higher than the control
(Fig. 1). In experiment 2, no
decrease in 13C values was observed after isolation for 3
days in treatments L–U and L–L compared to immediate dissection
(Fig. 2A). In treatment
L–L similar values were reported [t(15)=–0.12,
P>0.05], whereas in treatment L–U a significant increase was
observed [t(15)=–2.57, P<0.05].
When females were mated to males labelled in the larval stage and isolated
for 4 or 7 days after mating, similar 13C values were found
compared with immediate dissection (F2,26=2.23;
P>0.05; Fig. 2B).
It was also observed that females inseminated by 10-day-old males retained
similar amounts of label after isolation (II) as females inseminated by
4-day-old males, and 13C values after isolation were
comparable to immediate dissection (I) at both ages
(F3,38=1.12, P>0.05;
Fig. 2C).
Control samples
Spermathecae from females mated with males from the unlabelled treatments
in experiment 1 had similar 13C values as virgin females
[t(36)=–1.88, P>0.05] and the standards
[t(30)=–1.06, P>0.05]. The uninseminated females
from all treatments in experiment 2 had similar 13C values
as the virgin females [t(51)=–1.79, P>0.05].
Labelled versus unlabelled samples
The males from the labelled treatments in experiment 2 transferred
significantly high enough amounts of label to distinguish mean
13C values of inseminated spermathecae from uninseminated
ones (Fig. 2).
Threshold values
To determine the accuracy of the labelling, the threshold value in each
experiment was determined and is represented as a dotted horizontal line in
Figs 1 and
2. 13C values
of samples that appear above the threshold line are considered to have been
from females inseminated by labelled males, values below the line should
represent uninseminated females or females mated to unlabelled males. The
threshold value in experiment 1 was rather conservative due to some variation
in the samples from virgin females. Therefore, a small number of spermathecae
from the L–U treatment appeared below the threshold (4/21 samples).
Spermathecae from the dual labelling group appeared (except for one sample)
above the threshold value. However, a large proportion of spermathecae
inseminated by males labelled as adult only appeared below the threshold
value; hence this treatment was not considered optimal. In the second
experiment, males labelled in the larval and adult stage transferred enough
label so that the 13C value of each inseminated spermatheca
appeared above the 2 s.d. (standard deviation) threshold. Even if 3 s.d. was
used as a threshold value, all inseminated females had higher labels, and this
value is indicated in Fig.
2.
Between experiment variation
The mean (±s.e.m.) amount of carbon in the spiked samples differed
significantly between the experiments [t(268)=–26.32,
P<0.01]; in experiment 2, a higher amount of carbon was detected
(N=191, M=26.15±0.07 µg) compared to experiment 1
(N=79, M=22.84±0.11 µg). Between experiments,
13C values of control samples were similar for virgin
females [t(16)=0.62, P>0.05] and standards
[t(25)=0.99, P>0.05]. However, the males from experiment
1 transferred more label, resulting in higher 13C values,
than the males from equal treatments in experiment 3; L–U:
[t(7.50)=2.99, P<0.05], L–L: [t(20)=2.50,
P<0.05]. After isolation of the females for 3 days this difference
was no longer observed.
Males
The amount of label that was fixed in the reproductive system of males from
experiment 1 was similar to what was found in the females after mating. Larval
labelling resulted in a higher mean 13C value than adult
labelling and the labelling of both stages was superior over either singly
(
2: 18.12; P<0.01) (see Fig. S1 in supplementary
material). In atom%, this corresponds to a mean (±s.e.m.) enrichment of
2.52±0.46 atom% 13C for adult labelling (U–L),
4.64±0.67 atom% 13C for larval labelling (L–U) and
7.93±0.93 atom% 13C for labelling of both stages
(L–L). If males were sampled at an earlier interval (e.g. 4 days after
emergence), a similar amount of label was observed in treatment L–U
[t(10)=0.14, P>0.05], and a higher amount was observed in
treatment L–L [t(9)=2.36, P<0.05] compared to
sampling on day 11.
Life-history traits
Mating
Comparable insemination rates were found for labelled and control males in
all experiments (see Table S1 in supplementary material). The highest
insemination was observed in the first experiment when females were introduced
at the ratio of 2:1 (M:F). Experiment 2 aimed for a higher proportion of
uninseminated females, and insemination was between 57–84%, resulting in
adequate numbers of uninseminated females to validate the method.
Longevity
Labelling of males in the larval or larval and adult stage in experiment 2
had no detrimental effect on longevity; and a similar (2:
0.21; P>0.05) or even slightly higher (2: 9.27;
P<0.01) survival was observed as of control males
(Table 1). Similar observations
were made in experiment 3; longevity of males reared in trays with labelled
glucose was similar to that of the control males (2: 1.17;
P>0.05). Only in the second replicate was a lower longevity of
labelled males compared with the control observed (2: 11.65;
P<0.01). In both replicates, males from the unlabelled glucose
trays had significantly higher longevity than control males
(Table 1).
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Larval development and survival
The rate of pupation in trays where labelled or unlabelled glucose had been
added was similar to those without any glucose. Pupation started at day 7 and
continued until day 11, by which time the vast majority of L4 larvae had
pupated (data not shown). Larval survival was not affected by the addition of
[13C]-labelled glucose or unlabelled glucose to the trays; no
differences were observed between the three treatments
(F2,6=0.07; P>0.05)
(Table 2).
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Adult size
For each treatment, replicate and sex, wings were measured from 50
individuals that emerged on the second or third day of emergence (e.g. when
the majority of pupae emerged). No significant size differences were observed
in wings from both days, and data were pooled. Significant differences were
observed between replicates of the same treatment for males and females,
therefore data was analysed per replicate. For the females, size of the adults
was unaffected by the label; females emerging from trays with labelled glucose
or unlabelled glucose were similar in size (F2,146=1.47;
P>0.05) or larger (F2,151=5.09;
P<0.01) compared with females from the control trays
(Table 3). Some variation in
wing length between treatments and replicates was observed for the males. The
labelled trays produced the smallest (F2,148=9.09;
P<0.01) and the largest (F2,150=3.32;
P<0.05) males, but overall differences were small when compared to
the control males (Table
3).
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Sperm viability
No difference was observed in the hatch rate of the eggs from females mated
to labelled or control males, and the number of eggs laid was similar
(labelled: eggs=1786, hatch=0.81; control: eggs=2061, hatch=0.81).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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13C values than
uninseminated spermathecae and control samples, and the additional labelling
of the adult stage resulted in even higher 13C values
compared to larva labelling alone. Labelling in the adult stage alone was not
sufficient; it resulted in low amounts of label detected immediately after
mating, and the label seemed to diminish faster over time in spermathecae of
females isolated after mating. Somewhat similar results were observed in a
study with Aedes aegypti L., where it was found that when adult males
were offered radioactively labelled honeydew no labelled semen was transferred
to the females, even though the males themselves were highly labelled
(Dame and Schmidt, 1964).
Exposure in the larval stage did result in positive semen labelling and the
authors discussed that perhaps exposure during the early stages of
spermatogenesis, e.g. during the larval stage, was necessary to incorporate
the label in the semen (Dame and Schmidt,
1964). In our study, adult labelling did label the semen but in
low amounts, therefore emphasis was put on larval and larval plus adult
labelling treatments.
Mass spectrometric analyses of the males showed a highly enriched
reproductive system. The level of enrichment of the testis or the accessory
glands separately was not determined. Hence, we cannot specify the relative
contribution of the label in both fractions of the semen (e.g. spermatozoa or
accessory gland products). Although differential labelling may be of interest
at a later stage, for practical purposes and applicability of the method
developed, our current methods are considered adequate. The highest enrichment
that was estimated was 8.5 atom% 13C (e.g. sampled 4 days after
emergence, L–L treatment), whereas the target enrichment was 20 atom%
13C. Not all 13C added to the diet is recovered in the
mosquito, because label is lost as a result of respiration in the larval trays
and turnover in the insect. Respiration in the larval trays is brought about
by micro-organisms present in the water. When feeding, these micro-organisms
will incorporate 13C in their system, but due to respiration,
13C is also lost from the trays as 13CO2. As
a consequence of variations in the microbial fauna in larval environments and
the resulting levels of respiration, the amount of label in trays will vary,
and this could account for some of the variability observed in the amount of
label transferred by males between experiments. Mosquito larvae are
collector-filter feeders (Clements,
1992), and feed on dissolved particles in the water. The label
could have been ingested directly through the uptake of
[13C]glucose, or indirectly through the uptake of micro-organisms
that utilized the supplied larval diet
(Merritt et al., 1992). The
relative contribution of each pathway remains speculative at this stage.
For use of this technique in experimental settings, persistence of the
label in the spermathecae after mating is desirable, as females will not
always be dissected immediately after mating. It was observed that the label
was detectable in spermathecae up to 7 days after insemination, and
significantly higher 13C values than control samples were
reported. Moreover, unlabelled sugar feeding of males labelled in the larval
stage did not result in a traceable dilution of the semen label due to
turnover of 13C with 12C. Males up to 10 days of age
transferred similar amounts of label as younger males. An important finding,
because adult males and females replenish energy reserves by sugar feeding on
plant nectars in nature (Foster,
1995; Clements,
1999), and are maintained on sugar solutions in the
laboratory.
The threshold values used in the experiments were successful in classifying
labelled spermathecae from unlabelled samples. Threshold values were different
between experiments because 13C () values of the
reference standard, in our case virgin females, varied between experiments. In
experiment 1, some variation in the 13C values of virgin
females resulted in a somewhat conservative threshold, but in the subsequent
experiment, the triple standard deviation threshold value demonstrated with
99.7% confidence that all samples were classified correctly. The few outliers
in the dataset, especially observed in experiment 1, could not be attributed
to contamination during sample preparation. However, we cannot exclude a
possible contamination by other samples during drying, or perhaps
cross-contamination by the highly labelled males occurred. Even though the few
outliers were a cause for concern, in the subsequent experiment no such
outliers were observed and we are confident that they do not invalidate our
findings. Between experiments, the amount of carbon in the samples differed
significantly, even though the `spike' solution used in all experiments was
derived from the same stock and kept at 4°C. The amount of carbon in the
experimental samples is referenced to standard samples, which are slightly
different between experiments, causing these levels of inconsistency. However,
as the 13C value is a ratio and thus independent of the
amount of carbon in the sample, this variation has no impact on our findings.
The spiking of samples resulted in a dilution of the label and complicated
calculations of the actual 13C values; however, in our mass
spectrometry set-up this was necessary to raise the detection limit.
Nonetheless, the raw 13C values could effectively be used
for interpretation of results and data analysis.
We have established a proof of principle in the laboratory; and this
technique can be used to study a variety of issues related to mating in
anopheline mosquitoes, and other insects. Although we applied the label in the
aquatic stage, in insects lacking this stage, the label can be incorporated in
the larval diet, but the optimal treatment would need to be determined (e.g.
duration of labelling treatment, formulation and amount of labelling diet,
etc.). Besides laboratory-based studies, there is a great potential to use
this technique in the context of genetic control studies like the Sterile
Insect Technique, etc. The most important parameter in these studies is the
ability of released males to locate and inseminate wild females, and stable
isotopes can be used to determine which group of males was responsible for the
insemination. Preferably, these experiments take place in the field or in
large field cages (Knols et al.,
2002) to evaluate the insects in their natural environment
(Scott et al., 2002). Because
fitness of the labelled males is of high importance in these experiments,
impact of the label on a number of life-history traits was assessed. Exposure
to the label, even at the high quantities that were used, had no effect on
male mating ability. Longevity of labelled males was similar or higher than
the control in the first two replicates; in the last replicate, a somewhat
reduced longevity was observed, but in general longevity was high and well
beyond any life expectancy in a more natural situation. The labelling of
mosquitoes in the larval stage by adding glucose to the trays had no effect on
larval development; and the same result was observed in a study applying the
same technique but with lower amounts of labelled glucose added to the trays
(Hood-Nowotny et al., 2006).
Size of the females was not affected by the label, and although some variation
in the males was observed, overall differences were small. There is no impact
of the label on the ability of labelled sperm to inseminate eggs; and similar
results were reported when radioactive isotopes were used to label semen
(Young and Downe, 1978). As
such, stable isotope labelling meets most criteria of an `ideal marker' for
insects that should be durable, non-toxic, easily applied, does not impact on
the insects behaviour (e.g. growth, reproduction, life span), is clearly
identifiable, and inexpensive (Hagler and
Jackson, 2001). The latter could be contested in the case of
stable isotope analysis. Even though pricing of stable isotopes and sample
analysis have decreased over the last years
(Hood-Nowotny and Knols, in
press), it is still a relative expensive technique to use. 1 g of
[13C]glucose 99 atom% costs US $100, and 0.25 g was used per larval
tray. Sample analyses were done in-house but can be outsourced at a cost of $5
per sample (Hood-Nowotny and Knols, in
press). Another minor drawback of mass spectrometry is that the
sample analysis is destructive, leaving no possibility to repeatedly measure
samples.
In conclusion, larval labelling alone resulted in sufficient amount of label transferred to females to distinguish inseminated spermathecae from control samples, and the label persisted in spermathecae for at least 7 days after insemination. Males up to 10 days of age transfer similar amounts of label as younger males, indicating that larval labelling results in a life-long signature. The label had no influence on larval development and survival, longevity or mating ability and is therefore considered an ideal marker. The label is easy to apply, the sample preparation is straightforward and cost of sample analysis is reasonable. Although the technology presented was tested in anopheline mosquitoes, other candidate insects for genetic control studies, e.g. Aedes mosquitoes, fruitflies, tsetse flies, etc. are likely to benefit from the same technology. We believe that stable isotopes offer a great potential to study mating behaviour in insects. In addition, stable isotopes are environmentally safe and are thus likely to be well accepted both within the research community and by the public.
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Acknowledgments |
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Footnotes |
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References |
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