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Physiological Adaptations to Extreme Maternal and Allomaternal Care in Spiders

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Physiological Adaptations to Extreme Maternal and Allomaternal Care in Spiders

Abstract

Some semelparous species show terminal investment by suicidal offspring provisioning. This requires internal cellular disintegration for the production of regurgitated food and in preparation for the sacrifice of the female body to the offspring, however, we have limited insights into the extent and costs of such physiological modifications. Extreme provisioning is hypothesized to be limited to reproducing individuals because it requires physiological alterations triggered by reproduction. However, non-reproducing helpers-at-the-nest have been shown to engage in suicidal provisioning, prompting us to ask whether helpers undergo similar physiological alterations to brood provisioning as mothers, which would represent an adaptation to cooperative breeding. Using an experimental approach, we investigated the physiological consequences of extended maternal care in the solitary spider Stegodyphus lineatus and the cooperative breeder S. dumicola, and whether non-reproducing helpers (virgin allomothers) in S. dumicola show physiological adaptations to brood provisioning. To identify costs of offspring provisioning, we determined the energy expenditure (standard metabolic rate; SMR) and tissue disintegration over the course of brood care. In both species, brood care is associated with elevated SMR, which was highest in allomothers. Brood care results in progressive disintegration of midgut tissue, which also occurred in allomothers. On experimental offspring removal, these responses are reversible but only until the onset of regurgitation feeding, marking a physiological “point-of-no-return.” The mechanism underlying the onset of physiological responses is unknown, but based on our finding of mature eggs in mothers and allomothers, as opposed to the undeveloped eggs in virgins of the solitary species, we propose that oocyte maturation is a central adaptation in non-reproducing helpers to provide terminal allomaternal care.
ORIGINAL RESEARCH
published: 13 September 2019
doi: 10.3389/fevo.2019.00305
Frontiers in Ecology and Evolution | www.frontiersin.org 1September 2019 | Volume 7 | Article 305
Edited by:
James Luke Savage,
University of Sheffield,
United Kingdom
Reviewed by:
Per T. Smiseth,
University of Edinburgh,
United Kingdom
Anne Danielson-Francois,
University of Michigan–Dearborn,
United States
*Correspondence:
Trine Bilde
trine.bilde@bios.au.dk
Gabriele Uhl
gabriele.uhl@uni-greifswald.de
These authors share first authorship
These authors share
senior authorship
Specialty section:
This article was submitted to
Behavioral and Evolutionary Ecology,
a section of the journal
Frontiers in Ecology and Evolution
Received: 04 May 2019
Accepted: 30 July 2019
Published: 13 September 2019
Citation:
Junghanns A, Holm C, Schou MF,
Overgaard J, Malte H, Uhl G and
Bilde T (2019) Physiological
Adaptations to Extreme Maternal and
Allomaternal Care in Spiders.
Front. Ecol. Evol. 7:305.
doi: 10.3389/fevo.2019.00305
Physiological Adaptations to Extreme
Maternal and Allomaternal Care in
Spiders
Anja Junghanns 1†, Christina Holm 2† , Mads Fristrup Schou 2,3 , Johannes Overgaard 2,
Hans Malte 2, Gabriele Uhl 1
*and Trine Bilde 2
*
1General and Systematic Zoology, Zoological Institute and Museum, University of Greifswald, Greifswald, Germany,
2Department of Bioscience, Aarhus University, Aarhus, Denmark, 3Department of Biology, Lund University, Lund, Sweden
Some semelparous species show terminal investment by suicidal offspring provisioning.
This requires internal cellular disintegration for the production of regurgitated food
and in preparation for the sacrifice of the female body to the offspring, however, we
have limited insights into the extent and costs of such physiological modifications.
Extreme provisioning is hypothesized to be limited to reproducing individuals because
it requires physiological alterations triggered by reproduction. However, non-reproducing
helpers-at-the-nest have been shown to engage in suicidal provisioning, prompting us
to ask whether helpers undergo similar physiological alterations to brood provisioning
as mothers, which would represent an adaptation to cooperative breeding. Using an
experimental approach, we investigated the physiological consequences of extended
maternal care in the solitary spider Stegodyphus lineatus and the cooperative breeder
S. dumicola, and whether non-reproducing helpers (virgin allomothers) in S. dumicola
show physiological adaptations to brood provisioning. To identify costs of offspring
provisioning, we determined the energy expenditure (standard metabolic rate; SMR)
and tissue disintegration over the course of brood care. In both species, brood care
is associated with elevated SMR, which was highest in allomothers. Brood care results
in progressive disintegration of midgut tissue, which also occurred in allomothers. On
experimental offspring removal, these responses are reversible but only until the onset
of regurgitation feeding, marking a physiological “point-of-no-return.” The mechanism
underlying the onset of physiological responses is unknown, but based on our finding
of mature eggs in mothers and allomothers, as opposed to the undeveloped eggs in
virgins of the solitary species, we propose that oocyte maturation is a central adaptation
in non-reproducing helpers to provide terminal allomaternal care.
Keywords: brood-provisioning, allomaternal care, histology, physiology, semelparity, metabolic-rate, midgut
INTRODUCTION
Parental care involves a wide range of behavioral and physiological adaptations that increase the
fitness of a parent’s offspring (Trivers, 1972; Clutton-Brock, 1991; Royle et al., 2012). Parental care
is most commonly performed by females, and in the most extreme cases it involves regurgitation
feeding of the offspring for a prolonged period and matriphagy, in which the female sacrifices her
Junghanns et al. Physiological Responses to Brood Provisioning
body through cellular disintegration as a terminal investment in
her brood (Smiseth et al., 2012). The investment in parental care
relative to somatic maintenance or growth is strongly influenced
by life history and ecology (Stearns, 1992). In iteroparous species,
which reproduce more than once in their lifetime, trade-offs
between current and future reproduction are expected to lead to
progressive increase in reproductive effort with age as residual
reproductive value declines (Pianka, 1976; Clutton-Brock, 1991).
Semelparous species that reproduce only once are under selection
to allocate all available energy into their single brood (Stearns,
1992; Roff, 2002; Alonso-Alvarez and Velando, 2012), and this
terminal investment may favor major and potentially irreversible
physiological adaptions to increase the efficiency of maternal
care. Our knowledge of the physiology and plasticity of responses
associated with extreme brood provisioning is, however, limited.
This applies both to solitarily reproducing species, and perhaps
even more so for cooperative breeders, where non-reproducing
helpers also engage in extreme offspring care.
Cooperative breeders show reproductive division of labor,
where a few individuals produce the offspring and closely
related helpers take over some or all aspects of parental care.
The provision of extended brood care by non-reproducing
helpers is known from cooperatively breeding insects, spiders,
birds, and mammals (Wilson, 1971; Choe and Crespi, 1997;
Lubin and Bilde, 2007; Cant, 2012). The inclusive fitness
benefits obtained by helpers through their investment in
brood care may favor traits that increase the effectiveness of
alloparental care (Wilson, 1971; Creel et al., 1991; Adkins-
Regan, 2005; Montgomery et al., 2018). Such traits could
be physiological adaptations as for example thermoregulation
or regurgitation of nectar to produce honey by the worker
bee (Wilson, 1971; Choe and Crespi, 1997; Cant, 2012). A
particularly interesting question in the context of parental care
is whether direct offspring provisioning requires physiological
adaptations in non-reproducing helpers. For example, the
ability to lactate is expected to be triggered by hormones or
development of organs associated with reproduction (Patton and
Neville, 1997), and may therefore be limited to reproducing
individuals within the group. Interestingly, the ability to perform
spontaneous lactation in mongoose helpers is coupled with
pseudopregnancy, which indicates an adaptation to cooperative
breeding (Creel et al., 1991). Regurgitation feeding of the
offspring with previously digested food is also expected to require
special adaptations, and may depend on cellular degradation
of the gut tissue (Nawabi, 1974; Salomon et al., 2015). The
exhibition of physiological traits in non-reproducing helpers that
enable offspring provisioning by regurgitation feeding therefore
represents an adaptation to cooperative breeding, a hypothesis
that has not yet been investigated.
Spiders exhibit maternal care by wrapping their eggs in silk
cases and guarding the offspring (Foelix, 2011), or provisioning
the offspring with captured prey (Avilés, 1997; Lubin and
Bilde, 2007). Some species show extended care by performing
regurgitation feeding, i.e., females provide a nourishing fluid
for the offspring by regurgitation. This process is thought
to be an energetically demanding task that is accompanied
by physiological changes involving degradation of the midgut
(Nawabi, 1974; Salomon et al., 2015), which functions as a
storage organ for fat and glycogen (Alberti and Storch, 1983).
Several genera also show matriphagy, as females are consumed
by their offspring following the provisioning period (Kullmann,
1968; Toyama, 1999; Kim et al., 2000; Viera et al., 2007; Foelix,
2011). Here, we investigated the metabolic cost and physiological
consequences of reproduction and offspring provisioning in
two species of semelparous spiders of the genus Stegodyphus,
specifically in one solitary and one cooperatively breeding
species. In both species, mothers provide extended maternal
care including regurgitation feeding and matriphagy, and in
the social species also the helpers (allomothers) engage in
regurgitation feeding and are consumed by the offspring (Kraus
and Kraus, 1988; Lubin and Bilde, 2007). We hypothesized that
in both species, reproduction and regurgitation provisioning
are associated with an up-regulation of cost intensive processes
in relation to egg production and organ restructuring for
offspring care until matriphagy (Speakman and McQueenie,
1996; Vanfleteren and DeVreese, 1996; Ruhland et al., 2016;
Fowler and Williams, 2017), which can result in elevated standard
metabolic rate (SMR) (Barnes and Partridge, 2003; Metcalfe and
Alonso-Alvarez, 2010). We tested this prediction experimentally
by determining changes in SMR in response to oviposition
and regurgitation provisioning. In parallel, we investigated the
dynamics of internal morphological changes in response to brood
care, with focus on the midgut tissue as the primary storage
organ in spiders. We determined when structural changes of
the midgut occur during the reproductive cycle of both species.
Experimentally, we also examined whether physiological changes
to the midgut are permanent once the process has been initiated,
or reversible upon experimental removal of the eggs or offspring.
A female can produce a replacement clutch if she loses her brood
(Schneider and Lubin, 1997a; Futami and Akimoto, 2005; Viera
et al., 2007), but depending on when the brood is lost, there
might be a point of no return in the physiological dynamics of
offspring provisioning.
The cooperatively breeding Stegodyphus species show
reproductive skew in which up to 80 percent of females in a
nest are unmated (Salomon et al., 2008). Mothers as well as
female helpers provide extended maternal and allomaternal care
(Lubin and Bilde, 2007; Salomon and Lubin, 2007; Junghanns
et al., 2017). Since allomaternal care is provided by mated,
reproducing females as well as by unmated, non-reproducing
females (Junghanns et al., 2017), this warrants the question of
whether the evolution of allomaternal care by non-reproducing
helpers is associated with physiological adaptations that
trigger the ability to provide regurgitation feeding. Using the
cooperative S. dumicola, in which both mothers and allomothers
perform similar tasks and share the workload (Junghanns
et al., 2017), we examined if non-reproducing helpers exhibit
adaptations to offspring provisioning, and whether potential
changes in energy allocation patterns (SMR) and dynamics
of changes in the midgut tissue (histological examinations)
in response to offspring provisioning are permanent
or reversible.
The mechanisms that trigger the onset of physiological
preparations for regurgitation provisioning are not well
understood. If reproduction activates the ability to provide for
the offspring, this would support the hypothesis that mating
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Junghanns et al. Physiological Responses to Brood Provisioning
or oviposition initiates an internal maturation process that
physiologically enables mothers to provide regurgitation feeding
(Krafft and Horel, 1980; Feneron et al., 1996; Schal et al.,
1997; Schneider, 2002; Mas and Kolliker, 2008; Pinilla et al.,
2012). We investigated whether oocyte maturation is a proxy
for reproductive maturation and a prerequisite for the ability
to provide regurgitation feeding. In the solitarily breeding S.
lineatus, experimental cross-fostering previously revealed that
non-reproducing (virgin) females do not adopt and care for
cross-fostered brood (Schneider, 2002). In contrast to the solitary
species, however, non-reproducing helpers in the cooperatively
breeding Stegodyphus engage in all aspects of allomaternal care
(Junghanns et al., 2017), suggesting the evolution of adaptations
to offspring provisioning in non-reproducing helpers. This
ability may be triggered by oocyte development, as unmated S.
dumicola can produce unfertilized egg sacs (A. Junghanns and
C. Holm, pers. obs.), in contrast to unmated solitary S. lineatus
(Y. Lubin, J Schneider, and T. Bilde, pers. obs.). We propose that
reproductive maturation is a prerequisite for triggering extended
brood care in prospective allomothers, and predict that unmated
females undergo development of their reproductive organs in
preparation for brood provisioning as helpers. We investigated
this prediction by comparing oocyte maturation as a proxy for
brood provision ability between mothers and non-reproducers
of the solitary S. lineatus and mothers, non-reproducing helpers
and non-helpers of the cooperative S. dumicola.
MATERIALS AND METHODS
Study Species
The spider genus Stegodyphus (Eresidae) contains 20+species
(Kraus and Kraus, 1988; World Spider Catalog, 2018), most
of which are solitarily breeding, subsocial species that show
extended offspring care. Cooperative breeding has evolved
independently three times, suggesting that subsocial behavior
is the ancestral state (Johannesen et al., 2007; Settepani et al.,
2016). Females are semelparous, and mothers and helpers of the
social species provide extensive maternal care, in which offspring
are provisioned by regurgitation feeding and female self-sacrifice
(Lubin and Bilde, 2007). The solitary S. lineatus oviposits March-
June, and tends the egg sac for 30 days (Millot and Bourgin,
1942). Females provision the offspring with regurgitated fluids
and are consumed by their offspring about 2 weeks after
hatching (Schneider, 1995). The social spider S. dumicola lives in
communal nests, which arise from a single mated female and her
offspring (Lubin and Bilde, 2007; Settepani et al., 2017). Females
oviposit December-February, and mothers and allomothers care
cooperatively for the offspring for several months until they are
consumed by the offspring (Seibt and Wickler, 1987; Salomon
and Lubin, 2007).
Collection Sites and Animal Maintenance
Stegodyphus lineatus was collected in Israel in April 2012, from
dry water courses at two sites, Mt. Amasa (31.31N, 35.12E)
and Lehavim (31.36N, 34.83E), with a total number of 215
individuals. Females were collected before they matured to
adulthood and therefore prior to oviposition, to follow them
through their entire reproductive and maternal care period
(mothers), and to assure that we had virgin females available
(virgin controls). Mothers and virgin controls were kept within
their natural nest in individual plastic containers (90 ×70 mm),
at a constant temperature of 25C and a 12:12 h light:dark
period. Until oviposition, mothers were provided with a diet of
houseflies or crickets two-three times/week, after which feeding
was stopped as they do not forage during brood care (Schneider
et al., 2003). Virgin controls followed the same feeding scheme
and were not fed after mothers had oviposited.
The cooperative S. dumicola was collected in South Africa
during two consecutive summers before females matured. The
first collection took place in November 2013 at three sites,
Shingwedzi (22.98S, 31.30E), Middelfontein (24.68S, 28.55E),
and Mokopane (24.40S, 28.78E), where a total number of
24 nests was collected. The second collection took place in
November 2014 from two sites, Shingwedzi (22.98S, 31.30E)
and Skukuza (24.9S3, 31.69E), with four nests used for
histological analysis. To ensure virginity, subadult females were
separated from males and raised to adulthood. Some of the
mature females were paired overnight with a male from the same
nest. If traces of secretion were found on the females’ genital
openings the next day, she was considered mated (Junghanns
pers. obs.) and was then used as a mother in small experimental
colonies created for studying brood care. Unmated, adult females
were used as allomothers (helpers) and were grouped with a
mother from the same nest. In both seasons, virgin females
from laboratory colonies that contained unmated, non-helping
females (kept without males and reproducing females) were used
to assess potential internal changes in the absence of brood care
(virgin control).
In the first season, the experimental colonies of S. dumicola
contained 1-2 mated females and three allomothers with a
total number of 334 groups. All spiders were kept in a climate
chamber at 25C with a 13:11 h light:dark period. In the
second season, 21 colonies consisting of one mated female
and three allomothers were used for histological examinations.
Experimental and control colonies experienced a 12:12 light:dark
period and temperatures of 19C at night and 27C during
the day with a peak temperature of 30C for 2 h at noon.
Experimental colonies were kept in transparent plastic containers
(122 ×82 ×52 mm) with a plastic ring (diameter 53 mm) for silk
attachment. Control virgins were kept in hexagonal plastic boxes
(180 ×180 ×60 mm). All colonies were fed two to three times
per week during the entire experiment with a diet of houseflies
and crickets.
Experimental Design
The experimental design is outlined in Figure 1. First, we
assessed changes in SMR and morphology (midgut and ovaries)
in females at different stages during the natural brood care
period in both S. lineatus and S. dumicola (natural group).
Second, we determined the reversibility of physiological changes
by experimentally removing eggs or offspring from mothers
at different stages during the brood care period (removal
groups). Since in S. dumicola, all colonies contained mothers and
allomothers (non-reproducing helpers), we were at the same time
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Junghanns et al. Physiological Responses to Brood Provisioning
FIGURE 1 | Experimental design for examining physiological effects of brood care in females of the subsocial S. lineatus and cooperative S. dumicola. Upper box
(white): representative time frames in days (d) for the reproductive cycle in the solitary breeding S. lineatus (S. li) and the cooperative breeding S. dumicola (S. du).
Middle box (light/medium gray): treatment of the experimental females (S. li: mothers; S. du: mothers and non-reproducing helpers/allomothers). Natural groups (light
gray): SMR measurements or chemical fixation for histological analyses (H) took place after mating (Nm), oviposition (No), hatching of offspring (Nh), early in the
regurgitation period (Ner; S. li =5 days after hatch; S. du =6 days after hatch) and late in the regurgitation period (Nlr; S. li =10 days after hatch; S. du =24 days
after hatch). Removal groups (medium gray): egg sacs or offspring were removed at different stages (Ro/oviposition, Rh/hatching, Rer/early and Rlr/late regurgitation),
and females were kept alive until the time of expected matriphagy (S. li =15 days after hatching; S. du =31 days after hatching) and then chemically fixed for
histological analyses. SMR was measured at all following stages after experimental removal. Lower box (dark gray): virgin controls. In S. lineatus SMR of virgin controls
was measured whenever a mother from the natural group was measured and at expected matripaghy. Virgin controls of both species (virgin S. li and virgin,
non-helping S. du) were examined histologically in the beginning and the end of the experimental period.
able to investigate the effect of egg sacs and offspring removal
on allomothers at different stages. To assess physiological
changes that are not due to brood care, we established virgin
controls, i.e., virgin females that we followed over time. The
natural and removal groups in combination with virgin controls
enabled us to address the following questions: (1) Does extreme
brood care involve physiological changes in mothers? (2) Are
physiological changes during brood care reversible, and if so,
until which stage(s) during the brood care period? (3) Do
allomothers experience similar physiological changes as mothers?
(4) Is the ability to provide extreme brood care associated with
oocyte maturation?
All S. lineatus mothers and the experimental colonies of
S. dumicola were checked every day for oviposition. After
oviposition, individuals/colonies were randomly assigned either
to the natural group or to a removal group (Figure 1). The natural
group followed an undisturbed course of brood care and standard
metabolic rate (SMR) was measured at the following stages of
the females’ reproductive cycle: “Nm” mating, “No” oviposition,
“Nh” the day when offspring hatched (hatching), “Ner” midway
in the phase of regurgitation feeding (early), and “Nlr” end
of regurgitation feeding (late) (Figure 1, natural group). For
histology, some spiders from all groups (except for Nm and at
the time of matriphagy) were chemically fixed (Figure 1). SMR
measurements and chemical fixation was done 1 day after the
respective stage was reached.
The colonies in the four removal groups (Figure 1) were
manipulated by removing either the eggs or offspring at different
stages corresponding to those of the natural groups as explained
above (termed Ro, Rh, Rer, Rlr), to examine the effect of removal
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Junghanns et al. Physiological Responses to Brood Provisioning
on SMR (measured the day after removal), midgut morphology,
and oocyte stage. In all removal groups, females were maintained
until their expected death by matriphagy, at day 15 after hatching
of the spiderlings for S. lineatus (Schneider, 1995) and at day
31 for S. dumicola (Henschel et al., 1995; Reut Berger-Tal pers.
comm.). If a replacement clutch was laid, it was removed. At the
time of expected matriphagy the females were chemically fixed to
investigate midgut integrity and oocyte stage histologically.
Whenever SMR was measured in a S. lineatus mother of
the natural group, a virgin control was measured in parallel.
Mother and virgin control were matched as to the amount of
time that had passed from the beginning of the experiment
(Figure 1). In SMR measurements of S. dumicola, mothers and
allomothers from the same experimental colony were measured
simultaneously. For histology, virgin non-caring controls of both
species were sampled and chemically fixed to assess the state of
their midgut and ovaries in the beginning and at the end of the
experimental period. In S. lineatus, these virgin controls were
approximately between 60 and 70 days old (since maturation
to adulthood) when used for histology. This corresponds to
the age of senescence of reproducing females, destined to be
consumed by their offspring. Based on their age and on multiple
experiments raising females both under laboratory and semi-
natural conditions, we are quite confident that virgin S. lineatus
do not produce egg sacs. As virgin controls in S. dumicola
matured within colonies it was impossible to determine the exact
age of a female. However, S. dumicola virgins were on average
younger than S. lineatus virgins. This suggests that the pattern
of egg maturation (mature eggs in virgin S. dumicola, immature
eggs in virgin S. lineatus) is likely to be robust: despite the older
age of virgin S. lineatus they still had less developed ovaries.
Measuring Standard Metabolic Rate
Standard metabolic rate (SMR) was estimated from the rate
of CO2production (VCO2) by repeated measurements using
stop-flow respirometry (Lighton and Halsey, 2011) in a setup
as described by Jensen et al. (2014). Individual spiders were
randomly assigned to a measuring chamber (glass cylinder L:
9×D: 2 cm), which was held at a constant temperature of
25C with a 12:12 h light:dark period (S. lineatus) or 13:11 h
light:dark period (S. dumicola). To avoid desiccation, a piece
of filter paper (15 ×15 mm) with 0.25 ml solution of 2% agar
was added to each chamber. The system used two parallel 8
channel multiplexers (RM Gas Flow Multiplexer, Sable Systems,
Las Vegas, Nevada, USA) allowing for measurements of 16
parallel respirometry chambers that were measured sequentially
by opening and closing the chambers. These measurements were
repeated over a period between 18 and 24 h. Measurements were
obtained by flushing the chambers with CO2free air (washed in a
soda lime column, MERCK Millipore, Darmstadt, Germany) at a
fixed rate of 250 mL min1. The flow was controlled by a flow
meter (Side-Trak R
, Sierra Instruments, Monterey, California,
USA) and a flow controller (MFC 2-channel v. 1.0, Sable Systems,
Las Vegas, Nevada, USA). Each chamber was flushed every
30 min (S. lineatus) or 40 min (S. dumicola) resulting in 30–48
or 35 independent measurements of metabolic rate during the
entire measurement period. The first three measurements were
excluded to eliminate effects of stress from handling, and as these
spiders are very sedentary, we estimated SMR from the average of
the three lowest values obtained during the day of measurement.
This was done to gain the intrinsic metabolic rate and not the
total energy budget that would include phases of activity and
handling stress.
The rate of CO2production was calculated from the raw data,
with a script in Mathematica (version 7.0, Wolfram Research,
Champaign, Illinois, USA) by assessing a baseline for each CO2
peak and integrating the area below the curve. Any abnormalities
in the plot were discarded by manual checks. See further details in
Jensen et al. (2014). Data are reported as mass specific metabolic
rate (µL/min/g).
Statistical Analysis of SMR
SMR measurements were analyzed using general linear mixed
models (glmm) with Gaussian errors in the R package “lme4”
v. 1.1–15 (Bates et al., 2015). As the different removal groups
were initiated at different stages of the reproductive cycle, each
differed in the number of stages that followed removal (Figure 1).
For example, females that had their eggs removed at oviposition
(Ro) were measured four times from the stage of hatching to
matriphagy, while females that had their offspring removed at the
time of hatching (Rh) were measured three times from time of
early regurgitation to the time of expected matriphagy. For this
reason, we constructed separate models for each removal group.
In each model, the measurement from the removal group was
compared to the measurements from the respective stage of the
natural group (e.g., Ro compared to No). Due to limited data,
the late regurgitation stages were not statistically analyzed (Rlr
vs. Nlr, see Tables S2–S4 for details).
For S. lineatus, SMR comparisons were performed between
(1) the sequence of stages of the natural group and the virgin
controls, and (2) in separate comparisons of each removal group
stage with the corresponding natural group (oviposition: No vs.
Ro; hatching: Nh vs. Rh; early regurgitation: Ner vs. Rer; Table S2
for sample sizes). Each full model consisted of the fixed effect
natural group, removal group, or virgin control, stage across
the reproductive period (continuous variable, with the first stage
present in a model being 0 and subsequent stages 1, 2, 3. . . ),
and the interaction between group and stage. Hence, in case of a
significant interaction, model coefficients for the main fixed effect
of group refer to the difference between groups in the first stage
of the model. As spiders were measured multiple times across
and within stages, spider ID was included as a random effect. If
we identified a significant difference in SMR between a natural
and a removal groups, we subsequently compared this removal
group to the virgin controls. Accounting for multiple measures
by the inclusion of spider ID as a random effect caused some
structure in the residual plots. We identified this structure as
an overfitting of the individuals only measured once. To ensure
that this did not produce spurious significance, we validated all
significant p-values using subsets of the data. We did not find
any deviation between the original results and the results in the
validation (Table S1).
In S. dumicola, mothers and allomothers of the natural groups
(Tables S3, S4 for sample sizes) were compared by constructing
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Junghanns et al. Physiological Responses to Brood Provisioning
a glmm including stage of reproductive cycle (continuous as for
S. lineatus) and reproductive role (mother, allomothers) as fixed
effects as well as their interaction. The colonies were included
as random effects in all models of S. dumicola (see Text S1
for details). To investigate whether mothers and allomothers
were affected differently across the different removal groups, we
compared each removal group with the corresponding stage of
the natural group as was done for S. lineatus, however, with
the inclusion of an additional fixed effect differentiating mothers
from allomothers. The group comparisons performed were No
vs. Ro, Nh vs. Rh, and Ner vs. Rer. The full models for each
pairwise group comparison contained the fixed effects stage
(continuous as for S. lineatus), group and female role, and all
possible interactions.
For all models, significance of the highest order interaction
term was evaluated by comparing the full model with a reduced
model in which the highest order interaction term had been
omitted. If the interaction term was non-significant it was
omitted from the full model, which was then further reduced
to evaluate the significance of each of the lower order terms
and so forth. In case of a significant interaction, further model
reductions and significance testing of involved main effects
were halted. Models fulfilled assumptions of parametric analysis
unless noted and all model comparisons were performed with
likelihood ratio tests. All statistical analyses were performed in
R (R Development Core Team, 2018).
Histology
Natural groups: To assess morphological changes in the midgut
tissue during the natural course of brood care, brood caring
females were chemically fixed at four stages as in the SMR
analyses: No) at oviposition by the mothers, Nh) after hatching
of the offspring, Ner) in an early regurgitation phase and
Nlr) in a late regurgitation phase (Figure 1). To this aim, the
opisthosomata of 9 S. lineatus mothers, 14 S. dumicola mothers
and 35 S. dumicola allomothers (from 10 nests) were fixed on the
day or 1 day after the respective stages were reached.
Removal group: To investigate whether potential changes are
reversible, we investigated 11 S. lineatus mothers, 19 S. dumicola
mothers and 49 S. dumicola allomothers (from 10 nests) from
which eggs or offspring had been removed at the same life stages
as given above (Ro, Rh, Rer, Rlr). The females were maintained
until the expected date of matriphagy (see Figure 1 “removal
groups”) and then chemically fixed.
We additionally examined five virgin females of S. lineatus
and 14 unmated non-helping females (virgin control) from
seven nests of S. dumicola, the latter having been kept in
colonies consisting of unmated females only. All females were
anesthetized with CO2before their opisthosomata were separated
from the prosoma and the region around the spinnerets was cut
off to enable sufficient penetration of the tissue by the fixative.
The opisthosomata were chemically fixed in Duboscq-Brasil after
(Bouin, 1887) for at least 1 week. The samples were dehydrated
in an alcohol series, transferred to Tetrahydrofuran (THF) and
embedded in paraffin (Rotiplast). Five micrometers sections were
produced with a rotation microtome HM 360 and then stained
with AZAN (Geidies, 1954). AZAN stains basophilic structures
in red while acidophilic structures are stained blue. As a result,
the nuclei are stained red, connective tissue light blue, secretion
blue, and granules of the cells blue, red, or yellow (Burck, 1988).
Staining does not stain regurgitate only, but also other material
of similar biochemical properties. Thus, we focus on liquefied
(blue) material in the gut region. More coarse material was not
considered regurgitate but food remnants. The samples were
analyzed and photographed using an Olympus BX60 System
Microscope and Zeiss Axio Vision 4.8. To avoid interpretation
bias, the histological sections were analyzed blind with regard
to the identity of the samples. We categorized morphological
traits of the midgut tissue as correlates for changes during brood
care: the abundance of secretion granules (blue stained granules)
and the abundance of extracellular fluids, both of which are
considered to accumulate for regurgitation purposes (Nawabi,
1974; Salomon et al., 2015). Sample sizes are reported in Table S5.
RESULTS
Variation in SMR Over the Maternal Care
Period in S. lineatus
We found a significant and increasing difference in SMR between
mothers in the natural group and virgin controls over time,
with mothers showing higher SMR than virgins, illustrated by
a significant interaction term between stage and group (Table 1;
Figure 2A). This effect was mainly driven by a decrease in SMR
over time in virgin controls, while mothers in the natural group
had a stable SMR across reproductive stages (Table 1;Figure 2A).
We compared mothers of the four removal groups with the
mothers of the corresponding natural groups. In response to egg
removal, Ro mothers had a significantly and consistently lower
SMR than No mothers (Table 1,Figure 3). SMR of mothers in
the removal groups at the time of hatching (Rh) and of early
regurgitation (Rer) did not differ significantly from that of Nh
and Ner mothers (Table 1;Figure 3). These results suggest that
(1) mothers in the period where they provide offspring care
maintain a higher SMR than virgin females; and (2) the elevated
SMR during brood care is reversible: the SMR of mothers that
have their egg sac removed before hatching of the eggs steadily
returns to a state similar to that of a virgin female, while removal
of offspring after hatching does not cause a reduction in SMR
(compare Figure 2A and Figure 3).
Changes in SMR Over the Maternal Care
Period in S. dumicola
Comparing SMR between allomothers and mothers in the
natural groups revealed consistently higher SMR in allomothers
from the time of oviposition and onwards (Figure 2B). A
significant interaction term between stage and reproductive
role (allomother/mother) indicates that the disparity in SMR
of allomothers and mothers increased over time (Table 2;
Figure 2B). SMR in S. dumicola mothers showed a decreasing
trend over the brood care period while allomothers did not
(Table 1,Figure 2B). As in S. lineatus mothers, both mothers
and allomothers in S. dumicola showed a sharp decrease in SMR
after egg sac removal (compare Figure 3 and Figures 4A,B),
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Junghanns et al. Physiological Responses to Brood Provisioning
TABLE 1 | Statistical analyses of SMR in mothers of the natural and removal groups and virgin control females in the subsocial S. lineatus.
Group model Effect Estimate se χ2
(1) P
Natural group vs. virgin control Intercept 1.29 0.06 - -
Stage 0.03 0.03 - -
Group (Natural group, Virgin Control) Virgin Control 0.02 0.13 - -
Stage:Group 0.17 0.05 11.15 <0.001
Natural group Intercept 1.30 0.06 - -
Stage 0.03 0.03 0.87 0.352
Virgin control Intercept 1.26 0.12 - -
Stage 0.13 0.04 8.86 0.003
No vs. Ro Intercept 1.42 0.06 - -
Group (No, Ro) Ro 0.32 0.14 4.93 0.030
Stage - - 0.39 0.533
Stage:Group - - 0.28 0.597
Virgin control vs. Ro Intercept 0.83 0.04 - -
Group (Virgin Control, Ro) Ro 0.29 0.1 7.9 <0.001
Stage - - 0.91 0.339
Stage:Group - - 1.82 0.178
Nh vs. Rh Intercept 1.43 1.43 - -
Group (Nh, Rh) Rh - - 0.02 0.900
Stage - - 0.19 0.662
Stage:Group - - 0.24 0.623
Ner vs. Rer Intercept 1.38 1.38 - -
Group (Ner, Rer) Rer - - 0.01 0.940
Natural groups followed a natural brood care cycle and SMR was measured at different stages: No (oviposition), Nh (hatching of spiderlings), Ner (early regurgitation phase) and Nlr (late
regurgitation phase). Removal groups experienced the removal of offspring at one of the reproductive stages given above, termed Ro, Rh, Rer, and Rlr, and SMR was measured at the
time of removal and at every subsequent stage. SMR in virgin controls was measured whenever the SMR of a mother was measured (see Figure 1). Stage is a continuous variable of
the timepoint in the reproductive period (mating to late regurgitation). When a removal group is included in the model, stage lasts from the subsequent time point to late regurgitation
(Figure 3) as these are the possible time points for SMR assessments. In the case of a significant interaction between stage and group, further model reductions and significance
testing of stage and group were halted, and we tested the effect of stage on the groups in two separate models. For each group model the effect sizes from the minimal adequate
model are reported. Bold values are significant p-values.
suggesting that experimental removal of the egg sac resulted in
a significant reduction in SMR in both mothers and allomothers
compared to females in the natural groups (Table 2). After
hatchlings were removed, S. dumicola allomothers showed higher
SMR than mothers (Table 2, compare Figure 4A and Figure 4B).
There was no reduction in SMR compared to the females
from the natural group (Nh) (Table 2;Figures 4A,B). When
regurgitation feeding had begun, removal of offspring likewise
did not cause a significant reduction in SMR in mothers and
allomothers compared to females from the natural groups, but
SMR of allomothers was again higher compared to mothers
(Table 2;Figures 4A,B). Overall, this suggests for the social S.
dumicola that (1) allomothers exhibit a higher SMR than mothers
over the entire period of offspring care; and (2) the elevated SMR
during brood care is reversible both for mothers and allomothers
if the egg sac is removed.
Histology: General Results for Both
Species (see Also Tables S6–S9)
In the midgut, extracellular fluids that accumulate in lumina and
other extracellular spaces in preparation of regurgitation feeding
can be distinguished from food remnants by their structure: while
food remnants have a coarse and flaky structure and are stained
pinkish or gray (see for example Figure 8B), regurgitate appears
finely structured and stains appear blue after AZAN staining (see
for example Figure 8D).
We examined the ovaries of all females to determine the
developmental stages of the oocytes. The undeveloped ovaries
contain homogeneously structured pre-vitellogenic oocytes
(Trabalon et al., 1992), which appear pink in AZAN staining and
do not show larger granules (e.g., Figure 5A). Pre-vitellogenic
oocytes were smaller than 150 µm in S. lineatus and smaller
than 100 µm in S. dumicola. Maturing early (S. lineatus: up to
300 µm, S. dumicola: up to 170; e.g., Figures 8D,E) and late
vitellogenic oocytes (S. lineatus: up to 370 µm, S. dumicola:
up to 270; e.g., Figure 5B) are considerably larger and exhibit
a grained structure. As females with early or late vitellogenic
oocytes usually also contained less matured oocyte stages, we
classified them by the most mature oocyte stage found in the
ovaries (see also Tables S6–S9).
Morphological Changes in S. lineatus
Natural Groups
Virgin controls from the beginning of the experimental period
and the end of the experimental period did not differ in the
traits investigated. In virgin controls and natural mothers at
the stage of oviposition (No), blue stained secretion granules
were either absent, or only present in clusters and in low
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Junghanns et al. Physiological Responses to Brood Provisioning
FIGURE 2 | Data on Standard Metabolic Rate measured as VCO2(µL CO2/minute/gram) during the natural brood care period (natural group) in mothers and virgin
control females of the solitary spider S. lineatus (A), and in mothers and allomothers (non-reproducing helpers) of the cooperative spider S. dumicola (B). For each
measurement of mothers, simultaneous measurements were performed on virgin controls (S. lineatus) or allomother females (S. dumicola). The mothers succumbed
to matriphagy and were thus not measured at this stage. The VCO2measure provided is the average of the 3 lowest measurements out of 40. Error bars show the
standard error of the mean.
amounts (Figures 5A,6A), with the exception of one female
that was chemically fixed one day after oviposition and
showed massive amounts of secretion granules. In contrast,
in Nh mothers (hatching; Figure 6C) and Ner mothers (early
regurgitation; Figure 6E) secretion granules were common or
abundant. In mothers at the late regurgitation stage (Nlr), the
secretion granules were less frequent (Figure 6G) or absent.
In many females at Nh and onwards, large lacunae were
present, but mainly the middle parts of the midgut region.
These lacunae were filled with large amounts of dense and
finely structured extracellular fluids that often stained blue
(Figures 6C,E). The abundance of these fluids and their tendency
to stain blue was decreased at Nlr (compare Figures 6E,G). At
this time the size of the female’s opisthosomata was smaller
compared to earlier stages (compare scale bars Figures 6C,E
to Figure 6G). Interestingly, even in the late brood care stages
part of the midgut tissue stayed intact (Figure 6G). Lacunae
were never observed in virgin controls. These histological
investigations show that brood care is associated with progressive
tissue disintegration.
Morphological Changes in S. lineatus
Removal Groups
Stegodyphus lineatus mothers from which the egg sac had been
removed at oviposition (Ro) did not show secretion granules
in the midgut tissue (Figure 6B). When the offspring had been
removed at hatching (Rh) or early or late regurgitation (Rer,
Rlr), the abundance of secretion granules varied strongly. In
these stages, extracellular fluids were visible (Figures 6D,F,H),
similar to mothers from the corresponding natural groups.
However, in contrast to natural mothers, fluids often appeared
less dense and often did not stain blue. At Rh, the amount
of extracellular fluid found in the midgut of the mother was
low or very low (Figure 6D). The lacunae were small and often
diverticula did not show marked natural lumina. In Rer mothers,
the amount of extracellular fluids varied from high amounts
of extracellular fluids (Figure 6F) to none. Rlr mothers showed
comparable amounts of extracellular fluids as natural mothers
at Nlr (compare Figure 6G and Figure 6H). The comparison of
mothers from the natural groups and removal groups suggests
that disintegration of the midgut tissue is reversible until the
hatching stage and that the ability of mothers to reverse these
processes diminishes once regurgitation feeding has begun.
Morphological Changes in S. dumicola
Natural Groups
In the natural groups, we found similar progressive tissue
disintegration of the midgut along the brood care period in
both mothers and allomothers. Control virgins exanimated at the
beginning and the end of the experimental period did not differ
in the investigated traits (Figure 5B), as mothers and allomothers
at No (Figures 7A,B) never showed blue stained secretion
granules or accumulation of extracellular fluids (Figure 5B). In
contrast, the blue stained granules were abundant in mothers
and allomothers at Nh (Figures 7C,D). During the regurgitation
phase (Ner, Nlr), less secretion granules were found in mothers
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Junghanns et al. Physiological Responses to Brood Provisioning
FIGURE 3 | Data on Standard Metabolic Rate (SMR) measured as VCO2(µL
CO2/minute/gram) during the maternal care period in mothers of the solitary
spider S. lineatus. SMR of natural mothers (reproducing females that followed
a natural brood care cycle) was measured at mating, oviposition, hatching of
offspring, early regurgitation and late regurgitation. Natural mothers
succumbed to matriphagy and were thus not measured at this stage. In the
removal groups, mothers were measured after removal of the egg sac or
offspring at the same stages as well as at expected time of matriphagy. The
VCO2measure provided is the average of the 3 lowest measurements out of
40. Error bars show the standard error of the mean. The natural group data
presented here is the same data as presented in Figure 2A.
and allomothers (Figures 7E–H). Mothers of the natural group
showed small to middle sized lacunae with extracellular fluids in
the anterior parts of the midgut starting at Nh (Figures 7C,E,G),
and in allomothers from Ner onwards (Re in Figures 7F,H).
The lacunae never occurred in the posterior parts of the midgut
(Figure 7F), but sometimes lumina filled with extracellular fluids
were found in the mid-regions of the midgut (Figure 7H). In Nlr
females, only small amounts of extracellular fluids were observed
(Figure 7G and Re in Figure 7H).
The morphological changes in natural mothers and
allomothers of S. dumicola were similar to those in natural
S. lineatus mothers. However, as the size of the lacunae in S.
dumicola midgut tissue never reached the same extent as in S.
lineatus (compare Figures 6C,E with Figures 7C–F), changes in
S. dumicola appeared less pronounced. At Ner, the amount of
secretion granules was lower in S. dumicola than in S. lineatus
(compare Figure 6E with Figures 7E,F). However, at Nlr the
midgut of S. dumicola mothers and allomothers contained more
secretion granules compared to S. lineatus mothers at the same
stage (compare Figure 6G with Figures 7G,H).
Our histological investigations of S. dumicola showed that
the provisioning of extreme maternal care is associated with
progressive changes of the midgut tissue in both mothers and
allomothers, but changes in the midgut were less pronounced
than those of S. lineatus.
Morphological Changes in S. dumicola
Removal Groups
Rh mothers showed low amounts of secretion granules
and almost no extracellular fluids (Figure 8C). In contrast,
allomothers at the same stage showed middle-sized lacunae and
had accumulated extracellular fluids when they were scrutinized
(Figure 8D). At Rer and Rlr, both mothers and allomothers
showed high levels of secretion granules and medium to large
amounts of extracellular fluids which were present in natural
lumina of the diverticula, and middle-sized lacunae in the
anterior to the middle parts of the midgut (Figures 8E–H). The
comparison of natural S. dumicola females to the females of
the removal group shows that the amount of secretion granules
was lower in Rh compared to Nh (compare Figures 7C,D to
Figures 8C,D). However, compared to the natural groups Ner
and Nlr, the amount of extracellular fluids was higher in Rer
and Rlr mothers (compare Figures 8G,E to Figures 7G,E) and
in allomothers from the hatching stage (Rh) onwards (compare
Figures 8D,F,H to Figures 7D,F,H). These data suggest that in
mothers of S. dumicola, disintegration of the midgut tissue is
reversible until the hatching stage as in S. lineatus mothers. In
contrast, allomothers of S. dumicola were seemingly not able to
terminate and reverse processes when offspring were removed
at hatching.
Ovaries of S. lineatus and S. dumicola
The ovaries of virgin S. lineatus females did not differ between
individuals examined at the beginning and the end of the
experimental period and showed exclusively pre-vitellogenic
oocytes (Ov in Figure 5A and Table S6). Similarly, the ovaries
of natural mothers from all stages of brood care often contained
pre-vitellogenic oocytes (Ov in Figures 6A,C,G). In the removal
groups, most mothers exhibited late vitellogenic oocytes in their
ovaries at least in one stage (e.g., Ov in Figure 6B,H, see detailed
results in Table S6). These results suggest that mothers keep the
ability to mature oocytes if they lose their brood, even if this
happens late in the brood care period.
In S. dumicola, our data shows that allomothers as well
as virgin non-helping females are able to mature oocytes
in their ovaries. Virgin controls exhibited pre-vitellogenic,
early vitellogenic, or late vitellogenic oocytes (Figure 5B
and Table S7) in their ovaries. There was no significant
difference between virgin controls sampled at the beginning
and those sampled at the end of the experimental period.
The ovaries of natural mothers contained pre-vitellogenic
(Figure 7A) or early vitellogenic oocytes (Figure 7C), and
some exhibited late vitellogenic oocytes in their ovaries.
In contrast, natural allomothers of all stages had early
vitellogenic (Figure 7B) or late vitellogenic oocytes (Figure 7F)
in their ovaries. In the removal groups, late vitellogenic
(Figures 8A,G) and early vitellogenic oocytes (Figure 8E) was
present in most mothers. Allomothers from removal groups
showed early or late vitellogenic oocytes in most cases
(Figure 8D).
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Junghanns et al. Physiological Responses to Brood Provisioning
TABLE 2 | Statistical analyses of SMR in mothers and allomothers of the natural and removal groups in the social S. dumicola.
Group model Effect Estimate se χ2
(1) P
Natural group Intercept 1.57 0.04 - -
Stage 0.04 0.02 - -
Reproductive role (Allomother, Mother) Mother 0.05 0.04 - -
Stage:Reproductive role 0.08 0.02 12.77 <0.001
Natural group—Allomothers Intercept 1.58 0.05 - -
Stage 0.03 0.03 1.91 0.167
Natural group—Mothers Intercept 1.50 0.03 - -
Stage 0.04 0.02 3.67 0.056
Ro Intercept 1.63 0.04 - -
Reproductive role (Allomother, Mother) Mother 0.25 0.04 40.84 <0.001
Group (No, Ro) Ro 0.3 0.05 25.31 <0.001
Stage - - 1.66 0.198
Stage:Reproductive role - - 1.78 0.182
Stage:Group - - 1.87 0.171
Reproductive role:Group - - 0.94 0.333
Reproductive role:Group:Stage - - 2.21 0.138
Rh Intercept 1.62 0.05 - -
Reproductive role (Allomother, Mother) Mother 0.29 0.05 31.13 <0.001
Group (Nh, Rh) Rh 0.1 0.1 - -
Stage 0.05 0.07 - -
Group:Stage 0.35 0.12 8.97 0.003
Stage:Reproductive role - - 0 0.947
Reproductive role: Group - - 2.13 0.144
Reproductive role: Group:Stage - - 1.67 0.197
Rer Intercept 1.64 0.07 - -
Reproductive role (Allomother, Mother) Mother 0.28 0.09 10.12 0.002
Group (Ner, Rer) Rer - - 0.71 0.401
Reproductive role:Group - - 3.21 0.073
Natural groups followed a natural brood care cycle and SMR was measured at different stages: No (oviposition), Nh (hatching of spiderlings), Ner (early regurgitation phase), and Nlr (late
regurgitation phase). Removal groups experienced the removal of offspring at one of the reproductive stages given above, termed Ro, Rh, Rer, and Rlr, and SMR was measured at the
time of removal and at every subsequent stage (see Figure 1). Stage is a continuous variable of the timepoint in the reproductive period (mating to late regurgitation). When a removal
group is included in the model, stage lasts from the subsequent time point to late regurgitation (Figure 4) as these are the possible time points for SMR assessments. The model name
represents the pairwise comparison made within the different groups. For example, the model “Ro” compares effects of Ro and No between females of different reproductive roles
(mothers and allomothers). In the case of a significant interaction, further model reductions and significance testing of the involved main effects were halted. As Stage:Reproductive role
was significant in the model “natural group” we further investigated this by testing the effect of stage in allomothers and mothers in two separate models. For each group model the
effect sizes from the minimal adequate model are reported. Bold values are significant p-values.
DISCUSSION
Does Extreme Brood Care Lead to
Physiological Changes in Females?
We investigated the physiological response to maternal care in
spiders with a semelparous life history. We found an energetic
cost of brood care in the solitary S. lineatus, with the highest
SMR exhibited by mothers during the regurgitation-feeding
period. This is consistent with an up-regulation of energy
demanding processes associated with offspring care resulting in
elevated SMR (Barnes and Partridge, 2003; Metcalfe and Alonso-
Alvarez, 2010). Interestingly, in the cooperative S. dumicola we
found a consistently higher SMR of unmated helpers than that
of mothers. This can be interpreted as allomothers investing
relatively more in parental care than mothers do. A possible
explanation for this difference in investment is that allomothers
do not allocate resources to reproduction, but instead invest all of
their resources into the care of the brood, which represents their
entire reproductive fitness. Furthermore, S. dumicola mothers are
sometimes observed to produce an additional brood (Junghanns,
unpublished), for which resources may be preserved (discussed
further below).
The removal experiment strongly suggests that brood care
is correlated with an elevated SMR, as mothers of S. lineatus
as well as mothers and allomothers of S. dumicola experienced
a significant reduction in SMR after removal of the eggs.
Although regurgitation feeding has not yet begun at this stage,
histological data suggests that the transformation of midgut
tissue in preparation for provisioning of young is already initiated
when an egg sac is present. The ability to reduce SMR in the
removal experiment could indicate metabolic and morphological
plasticity as a strategy to save energy for a second reproductive
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FIGURE 4 | Standard Metabolic Rate given as VCO2(µL CO2/minute/gram) in cooperative S. dumicola mothers (A) and allomothers (non-reproducing, virgin
females) (B). Natural groups followed a natural brood care cycle. SMR of mothers and allomothers was measured at mating, oviposition, hatching of offspring, early
regurgitation and late regurgitation (same data as in Figure 2B). Females in the natural group succumbed to matriphagy and were thus not measured at this stage. In
the removal groups, SMR was measured after removal of the eggs (oviposition) or offspring at the same stages as given above and at expected time of matriphagy.
The VCO2measure provided is the average of the 3 lowest measurements out of 40. Error bars show the standard error of the mean.
FIGURE 5 | Midgut sections of virgin control females of Stegodyphus lineatus (A) and S. dumicola (B) sampled at the beginning of the experimental period. The
midgut tissue (MG) consists of diverticula embedded in storage tissue and surrounds the heart (H), reproductive organs (Ov) and the silk glands (SG). No blue stained
secretion granules or extracellular materials are visible. The ovary of S. lineatus contains exclusively pre-vitellogenic oocytes while the ovary from S. dumicola shows
far matured oocytes. Virgin Controls sampled at the end of the experimental period did not differ. Scale bars are 2,000 µm.
event in case the first brood is lost (Ricklefs and Wikelski, 2002).
We hypothesized that an increase in SMR reflects investment
in maternal care, but a decrease in SMR could instead reflect
energy allocated to egg production and a down regulation
of energy allocation to self-maintenance (Naya et al., 2007).
However, oviposition and brood provisioning in S. lineatus
mothers coincided with elevated SMR relative to virgin females,
consistent with our primary hypothesis.
We note that SMR of virgin S. lineatus females declined over
time. We do not have an explicit explanations for this pattern, but
to provide the most realistic comparison between virgin females
and mothers, who do not feed after oviposition, the virgin females
were also not fed after this point. This could explain the decline
in SMR compared to mothers. This is also consistent with the
observation that virgins do not mature eggs and never produce
an egg sac unless they are mated, and therefore do not need to
allocate energy to these processes. This result emphasizes that
reproducing females upregulate SMR in response to brood care.
In the solitary S. lineatus, the histological data support the
observed differences in metabolic rate between reproducing and
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Junghanns et al. Physiological Responses to Brood Provisioning
FIGURE 6 | Histological sections of opisthosomata of Stegodyphus lineatus mothers of the natural group (left column) and mothers of the removal group (right
column). Females were either chemically fixed (natural group) or the eggs sac or offspring was removed (removal group) at (A,B) oviposition (No, Ro); (C,D) hatching
of spiderlings (Nh, Rh); (E,F) early regurgitation phase (Ner, Re; 5 days after hatching); (G,H) late regurgitation phase (Nlr, Rlr; 10 days after hatching). Females in the
removal groups were chemically fixed 15 days after hatching of the offspring when matriphagy would occur under natural conditions. In the natural group by the time
offspring hatches (C), massive changes have occurred compared to virgin females or females at oviposition (A). Blue stained secretion granules are abundant,
and parts of the midgut tissue are dissolved with stained extracellular material (Re) accumulating especially in the mid-region of the midgut tissue while anterior parts stay
(Continued)
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Junghanns et al. Physiological Responses to Brood Provisioning
FIGURE 6 | intact for the longest time. At the late hatching phase almost no extracellular material (interpreted as regurgitant fluid) is left and the opisthosoma has
shriveled, but parts of the midgut tissue are still intact. When mothers were separated from offspring directly after hatching (D) they were able to terminate and reverse
processes, almost no extracellular material is visible. During regurgitation the ability to reverse the cellular disintegration diminishes as extracellular material remains.
Oocytes might mature in the removal group even until the late regurgitation phase. H, heart; MG, midgut; Mu, muscle; Ov, ovary; Re, extracellular material
(regurgitant); SG, silk gland. All scale bars are 2,000 µm.
virgin females, with disintegration of the midgut tissue and
accumulation of extracellular fluids occurring only in mothers.
This suggests that reproducing females undergo morphological
changes to meet the demands of regurgitation feeding with
liquefied body tissue (Kullmann, 1968; Nawabi, 1974; Salomon
et al., 2015). Accumulation of material for secretion, perhaps of
alkaline content (Burck, 1988), in preparation for regurgitation
feeding started at oviposition. By offspring hatching, the midgut
tissue of mothers formed large extracellular lacunae that indicate
ongoing disintegration of midgut tissue, and lacunae filled with
fine-grained fluids post-hatching most likely contain the fluids
that females will regurgitate to the young. During regurgitation,
females lose weight (Salomon et al., 2005), which is reflected by
reduced amounts of fluids in the late regurgitation stage of S.
lineatus and a smaller opisthosoma compared to earlier stages.
Morphological changes similar to those in S. lineatus but not as
comprehensive, were observed in mothers and allomothers of
the social S. dumicola. In S. dumicola, extracellular blue stained
fluids did not accumulate in high amounts in lacunae but were
often limited to natural lumina of the diverticula in the anterior
part of the midgut. The less dramatic changes of the midgut
tissue in the social S. dumicola are likely to reflect adjustment
of resource allocation to a longer maternal provisioning period,
as reproduction is not entirely synchronized within a nest, and
the provisioning period in social Stegodyphus is longer than that
of solitary congeners (Seibt and Wickler, 1988). The ability to
perform continuous allomaternal provisioning may also provide
an insurance against high female mortality (Jones and Riechert,
2008). Collectively, the data show that mothers and allomothers
undergo physiological changes in preparation for regurgitation
provisioning, which is initiated at oviposition.
Are Physiological Adaptations to Brood
Provisioning Reversible?
We examined whether physiological alterations are irreversible
once reproduction is initiated as an adaptation to a semelparous
life history, or whether some modulation is possible in case of
brood mortality. Our data indicate that physiological adaptations
to maternal care are reversible if the offspring are removed
early in the maternal care period. SMR decreased significantly in
mothers following egg removal, whereas removal of the offspring
at different times during the regurgitation period did not result
in a marked decrease in SMR. Histological examination showed
that mothers of both species, after removal of hatched offspring,
showed a reduced amount of secretion granules, and in S. lineatus
mothers extracellular fluids also diminished. This suggests that
mothers were able to terminate the production of secretion for
regurgitation and to reabsorb existing extracellular material using
the remaining intact diverticula. The implication could be that
mothers retain sufficient resources to produce a second clutch if
the first brood is lost early in the provisioning period (Schneider
and Lubin, 1997a), this is a common scenario as the risk of brood
loss to infanticidal males, predation, or parasitism in nature is
high (Schneider and Lubin, 1997a,b; Bilde et al., 2007). The ability
to produce a replacement clutch was confirmed by the presence
of late vitellogenic oocytes in the ovaries of some females in late
stages of regurgitation feeding. Indications of reversal processes
were less pronounced when removal of offspring occurred in the
regurgitation feeding period, at which point S. lineatus mothers
appeared unable to reabsorb regurgitation fluids. Similarly, in
S. dumicola, extracellular fluids accumulated when the offspring
had been removed during regurgitation, indicating that these
females were incapable of reabsorbing extracellular fluids, and
unable to stop the process of producing additional fluids. This
suggests that physiological plasticity in mothers of both species
diminishes from the onset of regurgitation, marking the time of
regurgitation feeding a physiological ‘point of no return’. The
life history of Stegodyphus, in which high mortality may have
favored semelparity, appears to be aligned with physiological
adaptations to extreme maternal care that are irreversible once
regurgitation feeding has begun. At this point, females may have
invested an amount of energy that reduces the likelihood that
their energy budget would meet the threshold for yet another
successful reproductive bout (Drent and Daan, 1980; Stearns,
1992). Intriguingly, as mentioned above, we have observed that
S. dumicola females can produce a second brood, but we need
a better understanding of the circumstances under which this
happens. The solitary S.lineatus, only produces a replacement
brood if they lose their first egg sac (Schneider and Lubin, 1997a),
and it is possible that the ability of S. dumicola females to produce
a second brood depends on the timing and relative investment in
the first brood.
Do Allomothers Experience Similar
Physiological Changes as Mothers?
In the solitary S. lineatus, maternal care behavior occurs
exclusively in reproducing females (Schneider, 2002), while
unmated helpers of the cooperative species engage in maternal
care (Salomon and Lubin, 2007; Junghanns et al., 2017),
suggesting that the physiological ability of unmated helpers to
provision the offspring is an adaptation to cooperative breeding.
Allomothers consistently experienced higher maintenance cost
measured as SMR compared to mothers, indicating that helpers
may even experience a higher cost of brood care than mothers.
This elevated cost could be explained by allomothers engaging
more frequently in prey capture, web building and other
tasks of colony maintenance in addition to food provisioning
(Junghanns et al., 2017). The histological examination confirmed
Frontiers in Ecology and Evolution | www.frontiersin.org 13 September 2019 | Volume 7 | Article 305
Junghanns et al. Physiological Responses to Brood Provisioning
FIGURE 7 | Histological sections of opisthosomata of Stegodyphus dumicola mothers (left column) and allomothers (right column) of the natural group that followed a
natural brood care cycle. Females were chemically fixed at (A,B) oviposition (No); (C,D) hatching of spiderlings (Nh); (E,F) early regurgitation phase (Ner; 6 days after
hatching); (G,H) late regurgitation phase (Nlr; 24 days after hatching). Mothers as well as allomothers show similar changes with blue stained granules accumulating in
the cells at the time of hatching (C,D). Extracellular material is only visible in small amounts often in anterior parts of the midgut tissue (e.g., E,F). Oocytes may undergo
maturation in mothers (C) and allomothers (F,H). H, heart; MG, midgut; Ov, ovary; Re, extracellular material (regurgitant); SG, silk gland. All scale bars are 2,000 µm.
that allomothers undergo similar changes in the midgut as
mothers. Already at the stage of hatching, allomothers appeared
unable to terminate and reverse these internal processes, instead,
they continuously accumulated extracellular fluids. This may
reflect that allomothers care for all offspring produced by
several mothers in the family group, i.e. in the event of
Frontiers in Ecology and Evolution | www.frontiersin.org 14 September 2019 | Volume 7 | Article 305
Junghanns et al. Physiological Responses to Brood Provisioning
FIGURE 8 | Histological sections of opisthosomata of Stegodyphus dumicola mothers (left column) and allomothers (right column) of the removal group of which egg
sacs or offspring had been removed. Offspring was removed at (A,B) oviposition (Ro); (C,D) hatching of spiderlings (Rh); (E,F) early regurgitation phase (Rer; 6 days
after hatching); (G,H) late regurgitation phase (Rlr; 24 days after hatching). Females were chemically fixed 31 days after hatching at the time matriphagy would occur
under natural conditions. Mothers and allomothers show almost no blue stained secretion granules when offspring were removed at time of hatching (C,D) compared
to females of the same stage in the natural group. Extracellular material (regurgitant) is accumulating more in mothers from early regurgitation and in allomothers from
hatching onwards than in control groups, suggesting the inability to terminate and reverse production of material for regurgitation feeding when offspring were
removed at hatching. Ovaries of mothers and allomothers frequently show late-vitellogenic oocytes (A–C,G,H). H, heart; MG, midgut; Ov, ovary; Re, extracellular
material (regurgitant); SG, silk gland. All scale bars are 2,000 µm.
Frontiers in Ecology and Evolution | www.frontiersin.org 15 September 2019 | Volume 7 | Article 305
Junghanns et al. Physiological Responses to Brood Provisioning
loss of one brood, there is still demand for allomaternal
provisioning. Since the unmated allomothers cannot invest their
resources in their own offspring, their reproductive fitness is
determined by inclusive fitness benefits from raising their sisters’
brood (Hamilton, 1964a,b; Smith and Wynneedwards, 1964).
The ability of unmated females to engage in suicidal brood
provisioning is therefore key for acquiring indirect benefits of
helping by kin selection, and likely represents an adaptation to
cooperative breeding.
Are Morphological Adaptations to Extreme
Brood Care Associated With Egg
Maturation?
The physiological capacity to engage in regurgitation feeding
may rely on an internal maturation process triggered by mating
or oviposition (Feneron et al., 1996; Mas and Kolliker, 2008;
Pinilla et al., 2012). Interestingly, allomothers and virgin non-
helping females of the cooperative S. dumicola showed early
and late vitellogenic oocytes in their ovaries—a mating event
does therefore not seem required for egg maturation. The
presence of late stage oocytes in their ovaries likely indicates
the physiological maturation process that precedes and triggers
regurgitation feeding. In ants, ovarian maturation of workers is
linked with the performance of certain tasks in the nest, with
nursing workers showing the most developed ovaries (Feneron
et al., 1996). The link between ovarian maturation and brood
care may have played a role in the evolution of cooperative
breeding within the genus Stegodyphus, since in the solitary
species S. lineatus, virgin females only contained pre-vitellogenic
oocytes, and did not oviposit or provide care when in contact
with spiderlings. We therefore suggest that maturation of ovaries
in allomothers of cooperative Stegodyphus is associated with the
onset of physiological preparations for brood provisioning and
represents an adaptation to engage in cooperative breeding.
In conclusion, the onset of reproduction triggers an increase in
standard metabolic rate (SMR) and causes structural changes in
the abdominal midgut tissue, indicating physiological responses
to regurgitation feeding with liquefied body tissue. Females are
able to terminate and partly reverse these internal morphological
changes until the start of regurgitation feeding, which marks
a physiological “point of no return.” Remarkably, allomothers
show similar or even stronger physiological response to brood
care than mothers. This could be an adaptation to continued
allomaternal care over prolonged periods in social nests, or it
could facilitate the production of a second brood by mothers, who
retain the ability to mature oocytes upon the loss of offspring. In
contrast to virgin females of the subsocial S. lineatus, unmated S.
dumicola allomothers often contained early and late vitellogenic
oocytes in their ovaries. This suggests that oocyte maturation is
a prerequisite for the onset of extreme brood care in unmated
helpers, and that oocyte maturation has shifted to an earlier stage
in ontogeny as an adaptation to cooperative breeding.
DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will
be made available by the authors, without undue reservation, to
any qualified researcher.
AUTHOR CONTRIBUTIONS
GU, TB, AJ, CH, and JO contributed to conceive the ideas and
designed the experiments. AJ and CH collected the data. JO and
HM supervised SMR data collection and analyses. GU supervised
histology. MS performed statistical analyses. TB, GU, AJ, and CH
wrote the manuscript.
FUNDING
The study was supported by the European Research Council
with ERC StG-2011-282163 to TB. CH was supported by Weis-
Fogh Foundation. JO was funded by The Danish Council of
Independent Research. AJ was supported by a stipend from
the federal state of Mecklenburg-Vorpommern, Germany and
received funding from the young academics and mobility
program of the International Office, University of Greifswald.
ACKNOWLEDGMENTS
We would like to thank Yael Lubin for hosting CH in Sede
Boger, Israel, and Michelle Greve for hosting CH and AJ in
Pretoria, South Africa. We thank Lena Grinsted for a steady
hand in preparing S. lineatus, and Bram Vanthournout for help
with measuring S. lineatus. We are indebted to everyone in The
Spider Lab Aarhus University and the Uhl lab at the University of
Greifswald for stimulating work environments.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fevo.
2019.00305/full#supplementary-material
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Conflict of Interest Statement: The authors declare that the research was
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Copyright © 2019 Junghanns, Holm, Schou, Overgaard, Malte, Uhl and Bilde. This
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Frontiers in Ecology and Evolution | www.frontiersin.org 18 September 2019 | Volume 7 | Article 305
... These spiders live in nests of 100-1000 of individuals (typically 85% female) and cooperate on web building, prey capture, feeding, and brood care . Mothers and non-reproducing female helpers (allomothers) practice a special form of regurgitation feeding, in which they feed the spiderlings a mixture of digested prey and dissolved intestinal lining (Junghanns et al., 2019), while males die off before the next generation emerges. When the spiderlings are old enough to begin capturing prey, they consume the adult females (matriphagy), and subsequently mate and reproduce with their siblings within the nest . ...
... In fact, social insects like ants, termites, bees, and bumblebees transmit and homogenize their gut microbiomes between colony members by fecal-oral transmission, trophallaxis, oral exchanges, and via the shared nest environment (Powell et al., 2014;Brune and Dietrich, 2015;Billiet et al., 2017;Zhukova et al., 2017). The high microbiome similarity between individuals from the same nest observed in the social spiders (Figure 7 and Supplementary Figure S1) may likewise be explained by continuous transmission and homogenization within the nest: Both (allo)maternal care and matriphagy (Junghanns et al., 2017(Junghanns et al., , 2019 are potential routes of pseudo-vertical, exclusively maternal, transmission (as males die off before spiderlings emerge), while communal feeding (Schneider and Bilde, 2008) and the shared nest environment may lead to horizontal transfer and homogenization of the microbiome. ...
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Social spiders have remarkably low species-wide genetic diversities, potentially increasing the relative importance of microbial symbionts for host fitness. Here we explore the bacterial microbiomes of three species of social Stegodyphus (S. dumicola, S. mimosarum, and S. sarasinorum), within and between populations, using 16S rRNA gene amplicon sequencing. The microbiomes of the three spider species were distinct but shared similarities in membership and structure. This included low overall diversity (Shannon index 0.5–1.7), strong dominance of single symbionts in individual spiders (McNaughton’s dominance index 0.68–0.93), and a core microbiome (>50% prevalence) consisting of 5–7 specific symbionts. The most abundant and prevalent symbionts were classified as Chlamydiales, Borrelia, and Mycoplasma, all representing novel, presumably Stegodyphus-specific lineages. Borrelia- and Mycoplasma-like symbionts were localized by fluorescence in situ hybridization (FISH) in the spider midgut. The microbiomes of individual spiders were highly similar within nests but often very different between nests from the same population, with only the microbiome of S. sarasinorum consistently reflecting host population structure. The weak population pattern in microbiome composition renders microbiome-facilitated local adaptation unlikely. However, the retention of specific symbionts across populations and species may indicate a recurrent acquisition from environmental vectors or an essential symbiotic contribution to spider phenotype.
... Mating occurs once a year, after which males die and all female colony members (allo-mothers) cooperate in caring for egg cases and hatchlings. At the end of the allomaternal care stage, juveniles feed on, and kill, the adult females in the colony (gerontophagy) (Seibt & Wickler 1987;Junghanns et al. 2017Junghanns et al. , 2019. This results in a colony structure with no overlapping generations, and all colony members are more or less synchronous with regards to age, developmental stage and body size Grinsted & Lubin 2019). ...
Article
Sociality in spiders has evolved independently multiple times, resulting in convergently evolved cooperative breeding and prey capture. In all social spiders, prey is captured by only a subset of group members and then shared with other, non-attacking group members. However, spiders' propensity to attack prey may differ among species due to species-specific trade-offs between risks, costs and benefits of prey capture involvement. We explored whether engagement in prey attack differs among three social Stegodyphus species, using orthopteran prey, and found substantial differences. Stegodyphus mimosarum Pavesi, 1883 had a low prey acceptance rate, was slow to attack prey, and engaged very few spiders in prey attack. In S. sarasinorum Karsch, 1892, prey acceptance was high, independently of prey size, but more spiders attacked when prey was small. While medium-sized prey had higher acceptance rate in S. dumicola Pocock, 1898, indicating a preference, the number of attackers was not affected by prey size. Our results suggest that the three species may have different cooperative prey capture strategies. In S. mimosarum and S. dumicola, whose geographical ranges overlap, these strategies may represent niche specialization, depending on whether their respective cautious and choosy approaches extend to other prey types than orthopterans, while S. sarasinorum may have a more opportunistic approach. We discuss factors that can affect social spiders' foraging strategy, such as prey availability, predation pressure, and efficiency of the communal web to ensnare prey. Future studies are required to investigate to which extent species-specific cooperative foraging strategies are shaped by ontogeny, group size, and plastic responses to environmental factors.
... Typically, basal maintenance costs have high priority in the energy allocation hierarchy because cellular and organismal homeostasis is essential for survival as well as a necessary (albeit not sufficient) prerequisite for the organism's ability to carry out other fitness-related functions. Some exceptions occur when basal maintenance is sacrificed for the benefit of other functions such reproduction in terminal breeders (Junghanns et al., 2019;Royle et al., 2012) or activity to escape an immediate threat (Haider et al., 2018;Haider et al., 2019;Husak et al., 2016). However, such priority reversals are not sustainable and eventually lead to mortality. ...
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Across several animal taxa, the evolution of sociality involves a suite of characteristics, a 'social syndrome', that includes cooperative breeding, reproductive skew, primary female biased sex-ratio, and the transition from outcrossing to inbreeding mating system, factors that are expected to reduce effective population size (Ne). This social syndrome may be favoured by short-term benefits but come with long-term costs, because the reduction in Ne amplifies loss of genetic diversity by genetic drift, ultimately restricting the potential of populations to respond to environmental change. To investigate the consequences of this social life form on genetic diversity, we used a comparative RAD-sequencing approach to estimate genome-wide diversity in spider species that differ in level of sociality, reproductive skew, and mating system. We analysed multiple populations of three independent sister-species pairs of social inbreeding and subsocial outcrossing Stegodyphus spiders, and a subsocial outgroup. Heterozygosity and within population diversity were 6-10 fold lower in social compared to subsocial species, and demographic modelling revealed a tenfold reduction in Ne of social populations. Species-wide genetic diversity depends on population divergence and the viability of genetic lineages. Population genomic patterns were consistent with high lineage turnover, which homogenizes the genetic structure that builds up between inbreeding populations, ultimately depleting genetic diversity at the species level. Indeed, species-wide genetic diversity of social species was 5-8 times lower than that of subsocial species. The repeated evolution of species with this social syndrome is associated with severe loss of genome-wide diversity, likely to limit their evolutionary potential. This article is protected by copyright. All rights reserved.
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