The effect of larval density on pre-imaginal development in two species of desert fleas.

Abstract

We studied the effect of density of larvae on pre-imaginal development in 2 flea species (Xenopsylla conformis and Xenopsylla ramesis) parasitic on 2 desert rodent species (Dipodillus dasyurus, adult body mass 20 g and Meriones crassus, 80 g). We predicted a decrease in duration of development with an increase in density of larvae. In general, in both flea species, duration of larva-to-pupa development decreased with an increasing larval density. In addition, this stage of development was longer in male fleas and in fleas from parents fed on D. dasyurus. The effect of larval density on larval development was manifested mainly when parent fleas fed on D. dasyurus. Duration of pupation decreased with increasing larval density only in offspring of fleas fed on G. dasyurus. In both fleas, pupation was longer in males. The effect of parent host on duration of pupation was found in X. ramesis only (longer if the host was M. crassus). Resistance of newly emerged fleas to starvation depended mainly on parent host species. Young X. conformis survived longer if their parents fed on D. dasyurus, whereas young X. ramesis survived longer if their parents fed on M. crassus. It was also found that (a) an individual flea that spent more time as a larva also spent more time as a pupa and (b) longer larval development resulted in a shorter time that a newly emerged flea was able to survive when starved.

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The effect of larval density on pre-imaginal development in
two species of desert fleas
I. S. KHOKHLOVA1, A. HOVHANYAN1,2, A. ALLAN DEGEN1and B. R. KRASNOV2*
1Wyler Department of Dryland Agriculture, French Associates Institute for Agriculture and Biotechnology of Drylands,
Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boqer Campus, 84990 Midreshet
Ben-Gurion, Israel
2Mitrani Department of Desert Ecology, Institute for Dryland Environmental Research, Jacob Blaustein Institutes for
Desert Research, Ben-Gurion University of the Negev, Sede Boqer Campus, 84990 Midreshet Ben-Gurion, Israel
(Received 12 April 2010; revised 9 May 2010; accepted 10 May 2010; first published online 12 July 2010)
SUMMARY
Westudiedtheeffectofdensityoflarvaeonpre-imaginaldevelopmentin2fleaspecies(XenopsyllaconformisandXenopsylla
ramesis) parasitic on 2 desert rodent species (Dipodillus dasyurus, adult body mass 20 g and Meriones crassus, 80 g). We
predicted a decrease in duration of development with an increase in density of larvae. In general, in both flea species,
duration of larva-to-pupa development decreased with an increasing larval density. In addition, this stage of development
was longer in male fleas and in fleas from parents fed on D. dasyurus. The effect of larval density on larval development was
manifested mainly when parent fleas fed on D. dasyurus. Duration of pupation decreased with increasing larval density only
in offspring of fleas fed on G. dasyurus. In both fleas, pupation was longer in males. The effect of parent host on duration of
pupation was found in X. ramesis only (longer if the host was M. crassus). Resistance of newly emerged fleas to starvation
depended mainly on parent host species. Young X. conformis survived longer if their parents fed on D. dasyurus, whereas
young X. ramesis survived longer if their parents fed on M. crassus. It was also found that (a) an individual flea that spent
more time as a larva also spent more time as a pupa and (b) longer larval development resulted in a shorter time that a newly
emerged flea was able to survive when starved.
Key words: density dependence, fleas, pre-imaginal development, rodents.
INTRODUCTION
Fleas (Insecta: Siphonaptera) are obligate haemato-
phagous parasitesmostabundant anddiverseinsmall
and medium burrowing mammals (see Medvedev
and Krasnov, 2006 and Krasnov, 2008 for reviews).
They alternate between periods when they occur on
the body of their host and when they occur in its
burrow or nest. In the majority of flea species, pre-
imaginal development is entirely off-host. With a few
exceptions, flea larvae are not parasitic and feed on
organic matter in the burrow and/or nest of the host.
The durationof development ofpre-imaginalfleas,
like that of all holometabolous insects, is highly
dependent on extrinsic environmental factors such as
ambient temperature,relativehumidityandsubstrate
structure (Margalit and Shulov, 1972; Silverman
et al. 1981; Silverman and Rust, 1983, 1985; Metzger
andRust,1997;Krasnovetal.2001a,b;Krasnovetal.
2002a,b). In addition, it may be affected by host-
related factors such as host species, body condition or
reproductive activity (Tripet and Richner, 1999;
Krasnov et al. 2004, 2005a). For example, Krasnov
et al. (2004) found that the length of time that larvae
of the rodent flea Xenopsylla ramesis took to hatch
depended on the host species exploited by parent
females. Development of eggs and larvae of X.
ramesis took longer when parent females were fed
on malnourished rodents (Krasnov et al. 2005a).
Tripet and Richner (1999) reported that the devel-
opment schedule of a hen flea Ceratophyllus gallinae
wasrelatedtothetimingand durationofthebreeding
period of its bird host.
Effects of extrinsic (thatis, environment- and host-
related) factors on flea pre-imaginal development
have proven to be physiologically based (Silverman,
1981; Silverman and Rust, 1985; Metzger and Rust,
1997; Krasnov et al. 2005a). Effects of intrinsic (that
is, flea-related) factors on their development have
received less attention. Nevertheless, Tripet and
Richner (2002) found that a high density of flea
larvae resulted in a decreased survival in C. gallinae,
although the duration of development was not
affected, whereas Krasnov et al. (2008) reported a
positive relationship between parent flea density and
duration of pre-imaginal development in 2 Xenop-
sylla species.
Some studies suggested that pre-imaginal fleas
may modify their developmental schedule (i.e. in-
crease or decrease duration of development) in a way
* Corresponding author: Mitrani Department of Desert
Ecology, Institute for Dryland Environmental Research,
Jacob Blaustein Institutes for Desert Research, Ben-
Gurion University of the Negev, Sede-Boqer Campus,
84990 Midreshet Ben-Gurion, Israel. Tel: +97286596841.
Fax: +97286596772. E-mail: krasnov@bgu.ac.il
1925
Parasitology (2010), 137, 1925–1935.
doi:10.1017/S0031182010000892
© Cambridge University Press 2010

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that maximizes their future survival. For example,
Tripet and Richner (1999) found that some pre-
imaginal C. gallinae traded off duration of develop-
ment againstsurvival. Bird hosts regularly clean their
nests and search forand kill larval fleas. The intensity
of cleaning behaviour is highest at the warmer centre
of the nest and less so at the cooler periphery. As a
result, larvae that cocoon at the nest periphery are
better able to avoid host anti-parasite behaviour, but
with the cost of delayed metamorphosis, i.e. longer
pupal stage. In another study, Tripet and Richner
(2002) reported that some newly emerged C. gallinae
dispersed very early from the site of emergence, even
prior to the nesting period of the bird hosts. Tripet
and Richner (2002) argued that pre-imaginal devel-
opment of these “early dispersers” took place after
hosts departed from their nests, i.e. under shortage of
food and independently of density. As a result, they
had not enough energy reserves for a prolonged
cocooned stage, so they had to emerge and initiate
their host search as early as possible. They shortened
their pupal stage and emerge early for the sake of
finding a host.
These results suggest that an increased or de-
creased duration of development may both be
advantageous for fleas, depending on ecological cir-
cumstances. For example, duration of pre-imaginal
development may depend on density of flea larvae
even when the larvae do not compete for food.
Indeed, the amount of organic matter in a host
burrow (where pre-imaginal development of fleas
takes place) may be high (remnants of host food, its
faeces etc.). However, even if fleas do not compete for
food as larvae, it is likely that they compete for host
blood as imagos, with this competition being often
mediated by host behavioural or immunological anti-
parasitic defences (e.g. Hawlena et al. 2007,2008). As
a result, fleas that emerge earlier may have an
advantage over fleas that emerge later. Consequently,
we hypothesized that flea larvae will respond to
increased larval density by changes in their develop-
ment schedule. We predicted a decrease in the
duration of larval and pupal development with an
increase in the density of larvae. Furthermore, this
effect may be mediated by the host because of the
aforementioned effect of host-related factors on pre-
imaginal development of fleas (e.g. host identity;
Krasnov et al. 2004).
We tested this prediction using 2 flea species,
Xenopsylla conformis Wagner and Xenopsylla ramesis
Rothschild, and 2 rodent host species, Dipodillus
dasyurus Wagner (adult body mass about 20 g)
and Meriones crassus Sundevall (adult body mass
about 80 g), all common in the Negev desert. The 2
rodents co-exist in various non-sandy and non-rocky
habitats, whereas the 2 flea species demonstrate
paratopic distribution (they occupy different, albeit
adjacent habitats with narrow zones of overlapping;
Krasnov et al. 1998). Despite somewhat different
environmental tolerance ranges within the habitats
of the 2 fleas, both species showed highest survival
and fastest development at similar air temperatures,
relative humidities and in the same substrate
(Krasnov et al. 2001a,b; 2002a,b). Both fleas attained
higher fecundity when they exploit M. crassus than
D. dasyurus (Krasnov et al. 2004).
MATERIALS AND METHODS
Fleas and rodents
We used rodents and fleas from our laboratory
colonies established in 1997 and 1999, respectively.
To guarantee genetic variability of rodent and flea
colonies, approximately 20 wild-captured rodents
and 200 wild-collected fleas were added to the
colonies yearly. Details on the maintenance and
breeding of rodents and fleas have been reported
earlier (e.g. Krasnov et al. 2001a,b; 2002a,b; 2004,
2007; Khokhlova et al. 2009). In brief, rodents were
maintained in plastic cages (60 by 50 by 40 cm or
20 cm) and offered millet seed and alfalfa (Medicago
sp.) leaves ad libitum. To obtain fleas, an individual
rodent host was placed in a cage that contained a steel
nest box with a screen floor and a pan containing a
mixture of sand and dried bovine blood (larvae
nutrient medium) on the bottom. This rodent was
infested with 10–15 (M. crassus) or 6–8 (D. dasyurus)
newly emerged fleas. Every 2 weeks, we collected all
substrate and bedding material from the cage and
transferred it into an incubator (FOC225E, Velp
Scientifica srl, Milano, Italy) where flea development
and emergence took place at 25°C and 75% relative
humidity.
Larvae
We fed 250 newly emerged adult females and 100
newly emerged adult males of X. conformis or
X. ramesis on adult male rodents (15–20 females
and 5–10 males per individual M. crassus and 8–10
females and 3–5 males per individual D. dasyurus)
for 2 h daily during 3 consecutive days. We used
male rodents because host gender affects feeding and
reproductive performance of fleas (Khokhlova et al.
2009). The numbers of fleas feeding simultaneously
on M. crassus and D. dasyurus differed because of the
size difference between the hosts. Consequently, the
number of fleas per unit body surface of a host was
approximately equal, so we assumed that parent fleas
feeding on either host experienced a similar degree of
competition. After feeding, fleas from each host were
placed in plastic cups (200 cm2), with a bottom
covered by a thin layer of sand and small pieces
of filter paper, transferred into an incubator and
maintained at 25 °C and 95% RH. Each feeding of a
flea was done on a different individual of the same
host species. Details on this procedure can be found
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elsewhere (Khokhlova et al. 2007; Krasnov et al.
2007).
After the third feeding, randomly chosen gravid
female fleas (200 fleas of each species) were placed
individually into 20 ml glass vials containing small
pieces of filter paper. Females oviposited on the
pieces of filter paper and eggs were transferred into
20 ml glass vials that contained a thin layer of clean
sand (8–10 eggs per vial). Vials were covered with a
5×5cmnylon screen held bya rubber band. The eggs
weremonitored twice daily until larvae hatched (after
6–7days;seeKrasnovetal.2001b).Experimentalfleas
and theireggs weremaintained at 25 °C and 95% RH.
Experimental design and procedures
For each flea species, 1000 larvae of the same age
(ca. 1 day old) were chosen randomly and transferred
in groups of 5, 15 and 30 individuals into 50 ml glass
vials(50 mmbottom diameter). Thevials, coveredby
perforated plastic lids and containing a 3 mm layer of
sand and a larval food medium (94% dry bovine
blood, 5% millet flour, and 1% ground excrements of
M. crassus or D. dasyurus), were placed in incubators
at 25°C and 95% RH. Larvae of the same species
from different females feeding on different indi-
viduals of the same host species were randomly
distributed among 3 treatments that differed in larval
density. The daily amount of food (larvae medium)
requiredfor successful development of flea larvaewas
determined earlier as 0·07±0·1mg per individual
larva (Krasnov et al. 2005b). The larvae were offered
this amount in our experiments and food medium
(0·07 mg per larva) was added daily to vials with
larvae, so that the food amount per larva was
equalized among and during treatments. Each treat-
ment was replicated 10 times totalling 120 exper-
iments (2 flea species×2 host species×3 densities×
10 replicates).
Vials with larvae were monitored twice daily until
all larvae pupated and either spun cocoons or died.
Cocoons were transferred into individual glass vials
with 1 mm of clean sand and covered by a nylon
screen (0·1 mm mesh) held by a rubber band. Vials
with cocoons were checked twice a day. They were
shaken slightly when checked, since vibration may
stimulate flea emergence. Vials with cocoons were
checked either until all adults emerged from their
cocoons or were considered dead. Vials with newly-
emerged adults were checked until all adults died.
After death of each imago, we identified its gender by
examination of its genitalia under light microscopy,
so that gender of each larva that survived until
emergence was known.
Data analyses
We estimated pupation and emergence success for
each group of larvae as the proportion of cocooned
larvae and the proportion of emerged adult fleas,
respectively. To evaluate the effect of larval density
on the duration of development and quality of newly
emerged fleas we calculated (a) time to pupation of
each larva; (b) time of pupation (from pupation to
emergence) of each pupa and (c) the longevity of each
newly emerged flea under starvation. This was done
only for individuals that survived from the larval
stage to emergence of the imago. We analysed the
effect of host species of parent fleas, larval density
and/or flea gender (independent variables) on pu-
pation and emergence success, development time of
larvae and pupae and time of death under starvation
of newly emerged fleas (dependent variables) using
2-way (for pupation and emergence success) or 3-way
ANOVAs (for remaining independent variables)
separately for X. conformis and X. ramesis. In ad-
dition, we tested whether (a) duration of larval
development affected duration of pupal development
and time to death under starvation and (b) whether
duration of pupal development affected time to death
under starvation taking into account flea gender,
parent host species and larval density. This was done
separately for X. conformis and X. ramesis using
Generalized Linear Models (GLM) with normal
distribution and log-link function. Thus, 2 models
were tested for each species. In one model, indepen-
dent variables were flea gender, parent host species,
larval density and duration of larval development,
whereasthedependent variablewasdurationofpupal
development. In another model, independent vari-
ables were those used in the previous model plus
duration of pupal development, whereas the depen-
dent variable was time of survival under starvation.
We searched for the best model separately for
X. conformis and X. ramesis using the Akaike’s
Information Criterion. We were interested in par-
ameter estimates of a model. Significance and sign of
these estimates for duration of a preceding time
period (i.e. larval and/or pupal development) would
indicate the occurrence of their negative or positive
effect on duration of a subsequent time period
(i.e. pupal development or time of survival under
starvation).
Variables were log- or arcsin (pupation and emerg-
ence success) transformed prior to analysis. Un-
transformed data are presented in the figures. Tukey’s
HSD tests were used for multiple comparisons.
RESULTS
In total, 77·1% larval X. conformis and 83·1% larval
X. ramesis survived until pupation, of which 87·8%
and 97·2%, respectively, survived until emergence
as new imagos. Survival of larval X. conformis did
not depend on either host species, larval density or
interaction between these two factors (F1,54=0·2,
F2,54=1·6 and F2,54=1·0, respectively; P>0·20 for
all) (Fig. 1a). Survival of larval X. ramesis also did
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Density and development in flea larvae

Page 4

not depend on either host species or larval density
(F1,54=0·02 and F2,54=2·6, respectively; P>0·08 for
both). However, interaction between these factors
was significant (F2,54=2·6, P<0·04) reflecting sig-
nificantly higher larval survival at the lowest density
but only when parent fleas fed on D. dasyurus
(Tukey’s HSD tests, P<0·05; Fig. 1a). In contrast,
pupal survival in X. conformis depended on both host
species and flea density (F1,54=5·5 and F2,54=10·0,
respectively; P<0·02 for both), but was not affected
by interaction between these factors (F2,54=1·5, P>
0·20). These effects were manifested in (a) signifi-
cantly higher survival if aparent hostwasD. dasyurus
than if a parent host was M. crassus (except for lowest
larval density) and (b) significantly lower survival
at higher density if a parent host was M. crassus
(Tukey’s HSD tests, P<0·05; Fig. 1b). Neither
factor or between-factor interaction affected pupal
survival in X. ramesis (F1,54=0·01, F2,54=0·8 and
F2,54=0·9, respectively; P>0·40 for all) (Fig. 1b).
Summary of ANOVAs of duration of larval and
pupal development of X. conformis and X. ramesis as
affected by flea gender, host species on which parent
fleas fed and larval density is presented in Table 1.
In X. conformis, duration of larval development
depended on all 3 factors, being longer (a) in males,
(b) in fleas born from parents fed on D. dasyurus
and (c) at lower larval densities (Tukey’s HSD tests,
P<0·05 for all; Fig. 2a). Duration of pupal develop-
ment of this flea differed between males and females
(being longer in males; Tukey’s HSD tests, P<0·05
for all), but did not generally depend on either parent
host species or larval density, except for females born
from parents fed on D. dasyurus (being longer at
lower larval density; Tukey’s HSD tests, P<0·05;
Fig. 2b). This explains the significance of two 2-way
interactions both involving flea gender (Table 1).
Duration of larval development of X. ramesis
depended on flea gender, parent host species and
flea density (Table 1), being longer in males, in larvae
produced by fleas fed on D. dasyurus and at lower
density (Tukey’s HSD tests, P<0·05; Fig. 3a).
However, the latter pattern occurred only in fleas
born from parents fed on D. dasyurus, explaining the
significance of host species×larval density inter-
action (Table 1, Fig. 3a). Pupal development of
this flea was longer in males and in fleas born from
parents fed on M. crassus. Male pupae produced
Fig. 1. Mean (±S.E.M.) proportion of (a) larvae that survived until pupation and (b) pupae that survived until emergence
in pre-imaginal fleas Xenopsylla conformis (Xc) and Xenopsylla ramesis (Xr) from parents fed on Meriones crassus (black
columns) or Dipodillus dasyurus (white columns) and maintained at different larval densities.
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Table 1. Summary of ANOVAs of the effect of flea gender, parent host species and larval density on
duration of larval and pupal development in Xenopsylla conformis and Xenopsylla ramesis
Flea
Period of
developmentFactor
D.F.
F
X. conformis
LarvalFlea gender
Parent host
Larval density
Flea gender
Parent host
Larval density
Flea gender×Parent host
Flea gender×Larval density
Flea gender
Parent host
Larval density
Parent host×Larval density
Flea gender
Parent host
Larval density
Flea gender×Larval density
1, 665
1, 665
2, 665
1, 665
1, 665
2, 665
2, 665
2, 665
1, 796
1, 796
2, 796
2, 796
1, 796
1, 796
2, 796
2, 665
15·0***
71·7***
24·8***
703·8***
2·7NS
0·2NS
7·1**
5·4**
4·9*
42·3***
3·7*
3·1*
1710·2***
40·5***
3·1*
6·2**
Pupal
X. ramesis
Larval
Pupal
* P<0·05.
** P<0·01.
*** P<0·001.
NS, Non-significant.
Only significant interactions are shown.
Fig. 2. Mean (±S.E.M.) duration of (a) larval and (b) pupal development in female (F) and male (M) Xenopsylla
conformis from parents fed on Meriones crassus (black columns) or Dipodillus dasyurus (white columns) and maintained
at different larval densities.
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by fleas fed on D. dasyurus developed significantly
longer at lower larval density, but no effect of larval
density on pupal development in female pupae or
male pupae produced by fleas fed on M. crassus was
found (Tukey’s HSD tests, P<0·05; Fig. 3b).
Results of ANOVAs of time to death under
starvation in newly emerged fleas are presented
in Table 2. In general, newly emerged female
X. conformis survived longer than male conspecifics
(Fig. 4a), although this was true mainly when their
parents fed on D. dasyurus (Tukey’s HSD tests, P<
0·05 if a parent host was D. dasyurus and P>0·05 if a
parent host was M. crassus). Both genders survived
longer if their parents fed on D. dasyurus than on
M. crassus(Tukey’s HSD tests, P<0·05;Fig. 4a). No
effect of larval density on time to death of newly
emerged X. conformis was found either in general or
in pair-wise within-gender and within-parent host
comparisons (Tukey’s HSD tests, P>0·05 for all).
Time to death of newly emerged X. ramesis was
affected by host species only (Table 2), being longer
in fleas from parents fed on M. crassus (Fig. 4b).
Best models describing relationships between
duration of larval development, duration of pupal
development and survival time under starvation
when all other factors were taken into account are
presented in Table 3. Signs of coefficients in these
models indicate positive correlation between dur-
ation of larval development and duration of pupal
development and negative correlation between dur-
ation of larval development and time of survival
under starvation. In other words, (a) an individual
flea that spent a short time as a larva also spent a short
time as a pupa and (b) longer larval development
resulted in a shorter time that a newly emerged flea
wasable to survivewhen starved. In addition, models
supported effects revealed in the ANOVAs (e.g.
longer duration of development of male as compared
to female pupae in X. conformis and longer time to
death of X. ramesis produced by parents fed on
M. crassus as compared to D. dasyurus).
DISCUSSION
In general, duration of flea development decreased
with an increase in larval density. Consequently, our
main prediction was supported. Moreover, mani-
festation and strength of the relationship between
Fig. 3. Mean (±S.E.M.) duration of (a) larval and (b) pupal development in female (F) and male (M) Xenopsylla
ramesis from parents fed on Meriones crassus (black columns) or Dipodillus dasyurus (white columns) and maintained
at different larval densities.
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duration of development and larval density was
mediated by host species and differed between flea
species and developmental stages.
Shorter development with an increase of larval density
Despite an equal food amount per larva among
treatments, larvae maintained at higher densities
pupated earlier and their pupae tended to shorten
the pupation period. This density-dependent res-
ponse suggests that intraspecific larval competition
occurs even when food does not represent a limiting
factor. Intraspecific competition for food among
insect larvae is well known (e.g. Nicholson, 1954;
Klomp, 1964; Beaver, 1974; Averill and Prokopy,
1987; Burrak et al. 2009). For example, Nicholson
(1954) described larval competition for food in the
sheep blowfly Lucilia cuprina and used this study to
introduce the concept of ‘scramble competition’
when all co-occurring individuals have equal access
to the limited resource. Moreover, a change of the
duration of development has been shown to be one of
the consequences of competition (Klomp, 1964).
The ultimate reason behind the decreased duration
of development of pre-imaginal fleas with an increase
in density may be associated with prospective intra-
specific competition among adult fleas. Increased
competition maydecreasefeeding, and consequently,
reproductive success of haematophagous arthropods
(Kelly and Thompson, 2000; see Khokhlova et al.
2007 and Krasnov et al. 2007 for fleas). A higher
density of flea larvae in a burrow of a host may result
in a higher density of adult insects that will compete
for blood from the same host individual, increasing
thus the severity of competition. This is especially
probable given that both M. crassus and D. dasyurus
are solitary and usually a burrow is occupied by a
single individual (Krasnovet al. 1996; Shenbrot et al.
1997; Gromov et al. 2000). As a result, under high
pre-imaginal density, individuals that will emerge as
imagoes earlier would more than likely have an
advantage over individuals that emerge later as later-
emerging fleas will more than likely feed on a host
under a higher density of co-exploiters than earlier-
emerging fleas. In other words, an earlier response of
larval fleasto high densities maybenefittheir survival
as adults. However,short durationof larval stagemay
negatively affect the viability of a pupa because an
early pupated larva may not have enough time to
accumulate energy reserves that will allow it to en-
dure desiccation during the pupal stage or to break
the wall of the cocoon and/or puparium (Silverman
and Rust, 1985). Furthermore, experiments of
Silverman and Rust (1985) suggested that flea emer-
gence is triggered when energy reserves drop below a
critical level. This was supported by the positive
correlation between durations of larval and pupal
stages in an individual flea in our study. In other
words, longer development should benefit a flea
larva. However, under high density, the larvae may
face a trade-off between its current success as a larva
and its future success as a new imago. Given this
trade-off, natural selection may favour a flexible
developmental schedule.
Proximate causesof
imaginal development are likely to include some
mechanisms that allow flea larvae to estimate the
density level. For example, flea larvae were able to
evaluate the amount of food available and respond by
remaining in or leaving the patch (Shryok and
Houseman, 2006). However, in our experiments the
amount of food available per larva was equal and,
thus, mechanisms other than direct food shortage
may play a role. For example, the density may be
indicated by the concentration of larval pheromones
or larval faeces. Although the existence of larval
pheromonehasneverbeen reportedforfleas,itiswell
known for other insect orders (Hartman et al. 1978
for Diptera; Deneubourg et al. 1990 for Coleoptera;
Jumean et al. 2005 for Lepidoptera). Larvae mayalso
respond to the number of encounters with each other
and/or frequency of tactile contacts. Another proxi-
mate cause could be variation in food selectivity
among individual larvae. In many flea species, the
most protein-rich part of the diet of larvae is faecal
pellets that adult female fleas expel near the clutch
(Cotton, 1970; Hinkle et al. 1991; Silverman and
Appel, 1994; Larsen, 1995; Hsu et al. 2002). These
pellets contain mainly blood of a host and, moreover,
their protein content is higher than that of the blood
upon which female fleas feed (Hinkle et al. 1991). In
ourexperiments, themostprotein-rich component of
the larval medium was dry bovine blood which has
been shown to be an adequate substitute of adult flea
faeces as a food source for flea larvae (Moser et al.
density-dependentpre-
Table 2. Summary of ANOVAs of the effect of flea
gender, parent host species and larval density on
survival time under starvation of newly emerged
Xenopsylla conformis and Xenopsylla ramesis
FleaFactor
D.F.
F
X. conformis
Flea gender
Parent host
Larval density
Flea gender
×Parent host
Flea gender×HS
×Larval density
Flea gender
Parent host
Larval density
1, 665
1, 665
2, 665
1, 665
6·1*
118·3***
2·0NS
4·6*
2, 6657·1**
X. ramesis
1, 796
1, 796
2, 796
0·1NS
8·7***
0·6NS
* P<0·05.
** P<0·01.
*** P<0·001.
NS, Non-significant.
Only significant interactions are shown.
1931
Density and development in flea larvae

Page 8

1991). Although the amount of this component
per larva was equal among treatments, its absolute
amount per group of larvae was obviously greater
in higher density treatments. If larvae vary in their
food selectivity, then highly selective individuals will
rapidly consume most of the dry blood, accumulate
the necessary amount of energy reserves and pupate
earlier. These explanations of the proximate causes of
density dependence of development require further
investigation.
Table 3. The best models explaining variance in duration of pupal development and survival time under
starvation as affected by flea gender, parent host species and duration of larval development in Xenopsylla
conformis and Xenopsylla ramesis and parameter estimates for these models
(AIC - Akaike’s Information Criterion, LR - likelihood ratio. Only significant coefficients are shown. Levels of effect of
categorical variables are ‘Dipodillus dasyurus’ for parent host species and ‘female’ for flea gender. All models are significant
(P<0·0001). See text for explanations.)
Flea
Independent
variableAIC LR χ2
Equation
X. conformis
Duration of pupal
development
Time of survival
−2090·0
−802·6
725·40·10+0·07*Larval development−0·06*Flea gender+0·01
*Parent host*Flea gender
0·23−0·15*Larval development+0·02*Flea gender+0·06
*Parent host
0·10+0·05*Larval development−0·01*Parent host
1·15−0·74*Larval development
196·1
X. ramesis
Duration of pupal
development
Time of survival
−2337·6
−639·7
27·4
136·8
Fig. 4. Mean (±S.E.M.) survival time under starvation in newly emerged (a) Xenopsylla conformis and (b) Xenopsylla
ramesis from parents fed on Meriones crassus (black columns) or Dipodillus dasyurus (white columns) and maintained at
different larval densities.
1932
I. S. Khokhlova and others

Page 9

Effects of flea species, flea gender and host species
The effect of density on larval development was
stronger in X. conformis than in X. ramesis, but the
oppositewasthecaseforpupaldevelopment.Inother
words, the between-flea difference in the magnitude
of the response to density was stage specific. This
may be associated with a different pattern of response
to a variety of extrinsic factors between larvae and
pupae of these fleas. Our earlier findings demon-
strated that both larvae and pupae of the 2 species
demonstrate species-specific sensitivity to the same
factor. For example, larval X. ramesis are more
sensitive to relative humidity than larval X. conformis
(Krasnov et al. 2001a), whereas pupal X. conformis
are more sensitive to air temperature than pupal
X. ramesis (Krasnov et al. 2001b). However, the
precise reason for differential stage-specificity in
sensitivity to larval density between the 2 flea species
is unknown.
In general, male fleas developed longer than female
fleas and offspring of fleas on D. dasyurus developed
longer than offspring of fleas on M. crassus. Gender
difference in the duration of development in fleas
has been reported for a number of flea species
(Sharif, 1949; Hudson and Prince, 1958; Vaughan
and Coombs, 1979; Amin et al. 1993; Metzger and
Rust, 1997; Kern et al. 1999; Krasnov et al. 2001b).
In the cat flea Ctenocephalides felis, it was suggested
that the biological significance of a shorter develop-
mentperiodbyfemalefleaswastopreventinbreeding
of fleas from the same cohort by increasing the
probability that females will mate with males from
other cohorts (Metzger and Rust, 1997). However,
in our earlier studies (Krasnovet al. 2001b), as well as
in this study, the time difference between male and
female emergence seemed to be too small for the
‘prevention-of-inbreeding’ explanation to be feas-
ible. Another reason for this pattern could be that
pre-imaginal flea males are more sensitive to micro-
climatic factors than pre-imaginal flea females
(Krasnov et al. 2001b).
Gender differences in the duration of development
might be a reason for fluctuating sex ratio in
populations of natural and laboratory reared fleas
(Bossard et al. 2000). For example, in laboratory
cultures of fleas it has been found that the peak of
emergence of one gender alternates with that of the
other gender, so that the snap-shots of young fleas
from laboratory cultures demonstrated either strong
male or strong female biases (Ma, 1993).
In our earlier study with the same flea and
host species (Krasnov et al. 2004), we found that
D. dasyurus as a host was inferior to M. crassus for
both flea species in terms of lower egg production
and/or slower egg development. This suggests that
exploitation of D. dasyurus as opposed to M. crassus
may result in the production of eggs of lower quality.
Longer post-egg development may, therefore, be
required to compensate for a lower quality of eggs.
This compensation, in turn, may result in a higher
amount of fat storage in newly emerged fleas that
allows them to survive longer under starvation.
However, this was found in X. conformis only,
whereas young X. ramesis survived longer if their
parents fed on M. crassus.
ACKNOWLEDGEMENTS
The experimental protocol used in this study met the
requirements ofthe 1994LawforthePreventionofCruelty
to Animals (Experiments on Animals) of the State of
Israel and was approved by the Ben-Gurion University
Committee for the Ethical Care and Use of Animals in
Experiments (License IL-27-9-2003). We thank an anony-
mous referee for helpful comments on the earlier version
of the manuscript. This study was supported by the Israel
Science Foundation (Grants no. 249/04 and 27/08 to
B.R.K., I.S.K and A.A.D.). This is publication no. 672
of the Mitrani Department of Desert Ecology.
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