Supplementary information
Measuring insect immune defences
The encapsulation response is an innate immune reaction of invertebrates that occurs
against a wide range of pathogens and parasites 1. It typically results in the formation
of multiple layers of dead melanised haemocytes 2, which isolate the parasite from the
haemocoel and kill it by asphyxiation or cytotoxic compound production 3. Melanin
deposition during the encapsulation response is most commonly initiated by the
haemocytes, but it can be activated by melanogenic enzymes (phenoloxidases)
circulating in the plasma 4. Melanin is a black pigment that is synthesised from the
amino acid tyrosine, a reaction that is in part catalysed by the enzyme phenoloxidase
(PO). It has been shown that this enzyme is essential in the melanisation of parasites 5
and it is the reactive intermediates of oxygen and nitrogen produced during
melanogenesis, that have been implicated in parasite death 4. In the mosquito
Anopheles gambiae, Gorman et al. 6 demonstrated that there is a shared genetic
mechanism for melanotic encapsulation of artificial implants and a real pathogen, and
previous studies have used the degree of darkening (melanisation) of artificial
implants as an assay of immunity e.g. 7,8,9,10. Introduction of an artificial nylon implant
in Atta colombica queens results in a darkening of the implant, consistent with
melanin production and possibly also involving haemocytes. In our 2004 samples, we
measured the degree of darkening of a nylon implant as our assay of immune
function, but in subsequent 2005 samples we also made haemocyte counts (see
below).
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Haemocytes are not only responsible for immune responses such as phagocytosis,
nodule formation and encapsulation, but also recognise foreign bodies in the
haemocoel and are intimately involved in wound healing 11. The total number of
circulating haemocytes has been found to be positively correlated with the ability to
encapsulate a parasitoid across six species of Drosophila 12,13, and to be genetically
correlated with encapsulation volume around a nylon implant 14. The number of
haemocytes in the the haemocoel has been frequently measured in the immune
literature e.g. 15,16,17 and is likely to reflect the capability of the immune system to deal
with pathogens, both in terms of the encapsulation response and because of
haemocytic involvement in other immunological processes. We may therefore expect
that if the haemocytes are utilised in the melanotic encapsulation response towards a
nylon implant that an increase in the encapsulation response (darker implant) would
be correlated with an increase in haemocyte numbers. Even if the haemocytes are not
involved in the encapsulation response, finding an increase in both encapsulation and
haemocyte numbers is likely to indicate a generally up-regulated immune system. We
therefore counted the number of haemocytes in a specific volume of haemolymph.
The encapsulation response and haemocyte number may also be influenced by other
physiological processes, for example mating can result in increased juvenile hormone
titers in insects 18,19,20. Juvenile hormone in turn has been found to have negative
effects upon the activity of PO 21, and because this enzyme is in part responsible for
the production of melanin in the encapsulation response we may see reduction in
encapsulation straight after mating. Furthermore, the developmental stage of the
insect can also cause variation in haemocyte numbers: for example, freshly emerged
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3
adult scorpion flies (Panorpa vulgaris) have lower haemocyte counts when compared
to larvae 22.
We therefore extended our data set in 2005 to test not only the extent to which our
immune assays could be reproduced across years, but also to see whether our
encapsulation findings would be reflected in the second immune correlate: haemocyte
numbers. To this end, on the 21st of May we marked another set of around 300
entrances of queen-burrows in four locations, all within 400 meters of one another and
with one location being the same as where the 2004 queens had been collected.
Furthermore, we sampled 36 winged virgin queens from four colonies in the direct
vicinity (on the 5th, 6th, 7th and 18th of May, respectively) to estimate whether the
encapsulation responses and haemocyte number before and after a mating flight
would be different. We expected that the encapsulation responses before the mating
flight would either be lower than those one day after mating (in the case that upregulation of the immune response triggered by solitary founding would already be
expressed as soon as queens no longer had grooming workers to protect them), or the
same (in the case that up-regulation of the immune response would take several days
to become effective). Similar to the previous year, one and nine days after the mating
flight we excavated 33 and 32 queens, respectively, from a random subset of the
marked burrows and placed them in individual petri dishes with moist cotton wool.
For each queen we made two immune measurements: encapsulation response and a
haemocyte count (per 0.01 µl haemolymph). Whilst digging we found that mortality
of founding queens was lower than in 2004, but it followed a similar pattern in both
years (Figure S1).
Percentage of queens found dead
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25
20
15
2004
2005
10
5
0
Day 1
Day 9
Days after mating
Figure S1. Mortality of founding queens of A. colombica in Gamboa, Panama one
and nine days after the mating flight for the years 2004 (black bars) and 2005 (open
bars).
For the haemocyte counts, each queen was chilled individually on ice and after she
had stopped moving (around 20 minutes later) she was put in the queen holder of the
adjusted machinery normally used for artificially inseminating bumblebees 23. A small
hole was made with a sterile syringe needle in the intersegmental membrane between
the 6th and 7th sternite. Through this hole 0.1 µl haemolymph was allowed to collect in
a calibrated glass capillary which had been pulled to a fine point. The haemolymph
was immediately added to 20 µl Ringer solution on a piece of parafilm and mixed
with the Ringer by sucking it up and down the capillary 10 times. For each queen, 2
Baer, Armitage & Boomsma: Cost of sperm storage
5
µl of this solution was added to a slide which had previously been coated with PolyD-Lysine. The slide was kept in a humid chamber overnight and then the droplet was
allowed to dry out. Up to two months later, the drops were rehydrated for 3-4 hours in
2 µl Ringer. Then 2 µl of DAPI solution was added to each drop and the slide was left
in a lightproof humid chamber overnight. The following day the excess DAPI was
removed by washing each drop six times with 2 µl Ringer solution. The haemocytes
were visualised and counted under a fluorescence microscope at 400x magnification,
to give the number of haemocytes in 0.01 l haemolymph.
Just after the haemolymph was removed, a piece of nylon was inserted into the same
hole to measure the encapsulation response, as in 2004. The queen was returned to her
petri dish and kept at ambient Panamanian temperature for 24 hours and then frozen at
–20°C, after which time the nylon was removed and photographed at 62.5x
magnification as described in the main paper. A digital camera (Canon EOS 350D)
connected to a Leica stereomicroscope was used. In both years, we took special
precautions to ensure that illumination conditions around the microscope and camera
were the same, by placing the equipment on the same light box and dimming ambient
light.
Baer, Armitage & Boomsma: Cost of sperm storage
80
90
**
Encapsulation
response
Haemocyte number
80
*
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
0
Before
1 Day
Haemocyte number per µl
haemolymph
Encapsulation response
90
6
9 Days
Time in relation to mating
flight
Figure S2. Encapsulation response and haemocyte numbers (means ± SE) for queens
of A. colombica sampled in 2005 before the mating flight, and one and nine days after
the mating flight. For both immune measures, mean values before the mating flight
and one day after the mating flight did not significantly differ, but values for one day
and nine days after the mating flight did significantly differ (*p = 0.022, **p = 0.005).
The results (Fig. S2) show that the encapsulation response and the haemocyte number
follow similar patterns across the two sampling times (three sampling dates). As some
of the analyses involved more than one measurement taken from the same queen (i.e.
encapsulation response and haemocyte number), we used a multivariate analysis of
covariance (MANCOVA) where appropriate. First, when comparing queens one day
and nine days after mating, there was no significant effect of weight (MANCOVA:
Wilks’ Lambda  = 0.952, F2,58 = 1.478, p = 0.237) or place of origin (the four
digging locations: MANCOVA: Wilks’ Lambda  = 0.917, F6,116 = 0.855, p = 0.530)
Baer, Armitage & Boomsma: Cost of sperm storage
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upon the combined dependent variables. These predictor variables were thus removed
from the analysis. In the remaining model, there was a significant effect of day of
digging upon the combined dependent variables encapsulation response and
haemocyte number (MANCOVA: Wilks’ Lambda  = 0.824, F2,62 = 6.618, p =
0.002). Subsequent univariate ANOVAs revealed that there was a significant increase
in both encapsulation response (F1,63 = 5.526, p = 0.022) and haemocyte number (F1,63
= 8.383, p = 0.005) after nine days (Fig. S2). Second, when comparing queens from
before the mating flight and one day after mating there was no significant effect of
weight (MANCOVA: Wilks’ Lambda  = 0.934, F2,65 = 2.31, p = 0.107) or time of
digging (MANCOVA: Wilks’ Lambda  = 0.951, F2,65 = 1.679, p = 0.195) upon the
combined dependent variables (Fig. S2).
Overall, the evidence is quite compelling that the immune responses measured are upregulated after mating and that it takes a number of days for this to happen. We
suggest that the build up of these additional defences most likely happens in response
to founding queens being exposed to soil microorganisms. This scenario would be
consistent with evidence collected for the mealworm beetle Tenebrio molitor. Moret
& Siva-Jothy 24 suggested that if an individual experiences a pathogen it may be
indicative of an increased risk of encountering pathogens in the future and that this
may help to explain why insects can produce immune responses that last for a long
time, the so-called ‘responsive-mode prophylaxis’. They found that T. molitor that
were pre-challenged with lipopolysaccharide prior to exposure to a fungal pathogen
had higher survival, compared to larvae that did not receive a pre-challenge. Thus we
may expect that if A. colombica queens have been exposed to pathogens in the soil,
they may have up-regulated their immune responses in such a prophylactic manner,
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and we suggest that it is this kind of increase we have measured after nine days. This
is consistent with a recent gene expression study in the fire ant Solenopsis invicta,
where it was shown that two precursors of antibacterial peptides were up regulated 24
hours after queens were inseminated 25.
The cost of mounting an innate immune response
One of the central ideas behind the study of the ecology of immune systems is that
their useage results in a cost for the host 26. In the most simplistic scenario, immune
costs can be classified into two categories, the first being the physiological costs of
maintaining and using the immune system, and the second the costs of having evolved
immunity in the first place 26. Life history theory predicts that when two traits
compete for allocation of materials and/or energy within a single organism a trade-off
will occur 27. Thus, if an immune response is costly in some way it will compete with
other energetically demanding processes that are occurring within the organism. We
therefore hypothesized that storing sperm and utilising the immune system are both
costly in A. colombica queens, and thus trade-off against one another for resources.
Evidence for the cost of using the immune system in invertebrates has been
accumulating by manipulation studies of specific aspects of the host biology and
measuring its effect upon immunity, predicting that individuals undertaking a costly
activity will show a reduced immune response e.g. 28, 29. Another fruitful approach has
been to challenge the immune system and to measure corresponding changes in other
traits, predicting that if producing an immune response is costly, an immune challenge
will negatively affect the other measured trait(s) e.g. 30, 31.
Estimating the number of males that contributed sperm
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We obtained 2-5 haplotypes from our spermatheca PCR’s, which matches the range
of number of fathers reported for A. colombica in Gamboa in a previous study 32,
where multiple paternity was inferred from offspring workers. A total of 15 alleles
were observed for the locus Etta 5-6TF and 16 alleles for the locus Etta 7-8TF. The
sums of the squared allele frequencies (Σp2) were 0.90 and 0.87, respectively. This
implies that our analysis underestimated the real number of fathers (1- Σp2) by 1013% for each marker locus, i.e. one of every ca. 8-10 inferred fathers was in fact two
fathers who had the same marker allele by chance, but most of these undetected males
were likely to be captured by the second locus 33. However, when alleles of fathers
were the same as queen alleles, a more conservative interpretation would be that these
were in fact maternal alleles (amplified from remnants of spermatheca tissue), so that
a lower number of fathers would be inferred. We believe that this problem has been
minor, because “allelic overlap” (a parental marker allele being shared by the queen
and her stored sperm) occurred in only 61 of the 391 (15.75%) single locus
comparisons of spermatheca-amplified alleles and queen alleles that could be made.
This overlap almost exclusively concerned the more common alleles, which are most
likely to be “sampled” twice by chance. Of the 18 Etta 5-6TF queen genotypes that
showed either double or single allelic overlap with spermatheca DNA, 13 (72.2%)
matched in only one of the two queen alleles (91% of the queens were heterozygous).
The corresponding figures for Etta 7-8TF were 28 cases of allelic overlap, of which
20 (69%) matched in only one of the two queen alleles (82.6% of the queens were
heterozygous). This is consistent with these single alleles representing a father allele
identical with a queen allele by chance, but not with the amplification of maternal
spermatheca tissue, as that should have recovered both maternal alleles from the
sperm sample. In only two cases we found complete overlap of maternal alleles with
Baer, Armitage & Boomsma: Cost of sperm storage
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paternal alleles (for both loci), and these queens were thus excluded from further
analysis, as their mate number remained ambiguous.
Statistics
All statistical analyses were performed using SPSS version 11 for Macintosh. Data
were tested for normality and homoscedasticity of variances using, respectively,
Kolmogorov-Smirnov tests and Levene’s test for equality of variances, and were
found to fulfill the conditions for parametric testing.
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