Vol.2, No.8, 855-859 (2010) Natural Science
Copyright © 2010 SciRes. OPEN ACCESS
Do male and female beetles (Tenebrio molitor) respond
differently to rat feces containing eggs from the
tapeworm, Hymenolepis diminuta?
John F. Shea
Department of Biology, Gonzaga University, Spokane, USA; shea@gonzaga.edu
Received 13 March 2010; revised 19 April 2010; accepted 23 April 2010.
Males and females often differ in their suscepti-
bility and exposure to infection. Thus, they may
also differ in their ability to avoid infection. The
beetle, Tenebrio molitor, risks infection with
cysticercoids when ingesting rat feces con-
taining eggs of the tapeworm, Hymenolepis di-
minuta. Previous studies demonstrated that
beetles sometimes prefer infective feces sug-
gesting that the tapeworm influences the for-
aging behavior of beetles. After recording beetle
movement in an arena containing infective and
uninfective feces for one hour, sex main effects
were not significant, but the effect of sex asked
in trials was significant for males. Specifically,
more male beetles occurred on the uninfective
bait than on the infective bait. This observation
suggests that male beetles avoid infective feces,
which decreases their probability of infection
with H. diminuta. If the cost of infection is higher
in males, then selection to avoid infective feces
may act more strongly on males.
Keywords: Hymenolepis diminuta; Cysticercoid;
Tenebrio molitor; Behavior; Coprophagy; Parasite
In general, male vertebrates tend to show a greater in-
tensity and prevalence of parasites than females [1-3],
while the same pattern is not observed as commonly in
invertebrates [4]. However, there are some exceptions in
which experimentally infected arthropods result in a
male infection bias [5-7]. This male infection bias in
invertebrates may be linked to differences in infection
susceptibility that are immunological [8-9], differences
in infection exposure that are behavioral [10-11], or
some combination [12]. If susceptibility and exposure to
infection differs by sex, then does avoidance of infection
also differ by sex? The beetle-tapeworm system lends
itself to the study of this question.
In the tapeworm Hymenolepis diminuta, adults live in
the small intestines of rodents, the definitive host. Eggs
pass out of the rodents in their feces. The cysticercoid, or
larval stage, occurs in an insect (the intermediate host,
most often a beetle) that becomes infected by feeding
upon infective feces. The rodent completes the cycle
when it ingests an infected beetle. Thus, the success of
this parasite depends on rodent-to-beetle transmission,
which depends upon consumption of the egg stage by
the beetle.
Prior studies [13,14] tested the fecal preference of
groups of beetles, but did not examine unisex groups of
beetles. One study [15] tested the fecal preference of
tapeworm-infected male and female beetles (Tenebrio
molitor) both in groups and as individuals. Only one
study [5] examined unisex groups of uninfected beetles
(T. molitor), which allows for sex comparisons while
eliminating potentially confounding interactions between
the sexes during the preference trial. The purpose of this
study is to assess the relative importance of sex in fecal
preference by recording beetle movement in an arena
containing infective and uninfective feces at one-minute
intervals for one hour.
The “OSU Strain” [16] of H. diminuta was maintained in
male Sprague-Dawley rats and beetles (T. molitor).
Three rats were infected with 30 cysticercoids and
maintained on commercial rodent chow and water. Three
additional rats, obtained from the same commercial
source, of identical age and from the same litter, were
maintained under the same conditions as the infected rats
to serve as the source of control (uninfective) feces. On
the morning of the trials, rat cages were checked every
10 minutes, and fecal pellets were collected with forceps
J. F. Shea / Natural Science 2 (2010) 855-859
Copyright © 2010 SciRes. OPEN ACCESS
to minimize contamination by rat urine. After each trial,
the infective fecal pellets were examined to verify the
presence of H. diminuta eggs.
Beetles were maintained on wheat bran, and small
pieces of potato were added to the cultures on a regular
basis. Pupae were removed from the cultures, and male
and female pupae [17] were placed in separate dishes
containing wheat bran. Beetles that emerged during a 24 h
period were collected such that a daily cohort of beetles
was maintained for both sexes.
Before the trial, seven to ten day-old male and female
beetles (starved for two to three days) were placed in
unisex groups of ten. To mitigate pseudo-replication,
twelve groups of each sex were used in the preference
trial in an alternating sequence. Each group was placed
under a glass bowl in the center of a plastic arena under
red light conditions. Beetles were provided with two
types of bait: uninfective (control) or infective feces both
of which had been collected within 10 minutes of defe-
cation and randomly paired. Control feces were posi-
tioned randomly either in areas 1 and 3 or 2 and 4 (Fig-
ure 1) and infective feces were positioned in the oppos-
ing areas. A paper towel was used to mash the fecal pel-
lets on the squares to prevent their displacement by bee-
tles during the trial. Each bait area (25 mm2) was large
enough for all beetles to occur simultaneously and was
located in a quadrant containing no bait (defined as a
non-bait area). After 15 minutes, the bowl was removed
and the beetles’ movements were videotaped for 60 min-
utes under red light conditions. During video play back,
the number of beetles observed at each bait area (square)
and non-bait area (quadrant) was recorded at one-minute
intervals. These occurrences were then summed over the
60 minute trial to obtain the total number of beetle oc-
currences at infective and control baits as well as
non-bait areas.
Preference was measured as the difference between
the number of beetles occurring at infective and control
baits at each minute of the 60 minute trial. The same
calculation was performed for the number of beetles
occurring in non-bait areas. These differences in the
number of beetle occurrences in the bait and non-bait
areas were analyzed with the Mixed procedure in SAS
(v.8, Cary, NC) with sex and trial as class variables and
sex nested in trial as a random effect. The response
variable was defined as the number of beetles at control
bait/non-bait minus the number of beetles at infected
bait/non-bait. Thus, a difference of zero indicates no
The maximum proportion of female and male beetles
occurring at the bait areas for each 60-minute trial was
plotted against the average proportion of beetles at the
bait areas (after an arcsine square root transformation for
both proportions), and the slopes were compared. The
same was done for the maximum proportion of female
and male beetles occurring in the non-bait areas.
The number of beetle occurrences did not differ between
males and females in bait (Table 1; Mixed procedure, P
= 0.159) or non-bait areas (P = 0.873). However, more
males occurred at the uninfective bait than at the infec-
tive bait (X ± SE = 1.58 ± 0.54, P = 0.008), while fe-
males did not differ in the number of occurrences at ei-
ther bait (X ± SE = 0.46 ± 0.54, P = 0.412). The number
of beetle occurrences in the non-bait areas did not differ
between infected and control halves of the arena for
males (Table 1; X ± SE = 0.30 ± 0.28, P = 0.292) or for
females (X ± SE = 0.24 ± 0.28, P = 0.403).
In a plot of the maximum number of beetles at each
bait area against the average number of beetles at each
bait area (data from Table 2), the slopes did not differ
between males and females at the infective (F1, 20 =
0.045, P = 0.834) or the control bait areas (F1, 20 = 2.75,
P = 0.113). In a plot of the maximum number of beetles
in each non-bait area against the average number of bee-
tles in each non-bait area (data from Table 3), the slopes
Figure 1. Diagram of the test arena for groups of beetles. Al-
ternative baits were placed in the square bait areas located in
four quadrants. Position of the control feces was determined
randomly (e.g., either squares 1 and 3, or 2 and 4). Each bait
area was 25 mm2 and located in a quadrant containing no bait
defined as a non-bait area. The diameter of the center circle
was 64 mm and the distance from its center to the edge of a
square was 75 mm.
J. F. Shea / Natural Science 2 (2010) 855-859
Copyright © 2010 SciRes. OPEN ACCESS
Table 1. Results of the mixed procedure (SAS v.8) for beetle preference by sex for the bait area (top), and for the non-bait (beetles in
quadrant–beetles on bait) area (bottom) in group trials.
Effect Mean difference estimate1,2 s.e. t p
Sex -1.12 0.77 -1.46 0.159
Trial (males) 1.58 0.54 2.90 0.008
Trial (females) 0.46 0.54 0.84 0.412
Sex -0.06 0.40 -0.16 0.873
Trial (males) 0.30 0.28 1.08 0.292
Trial (females) 0.24 0.28 0.85 0.403
1For males and females, the mean difference equals total number of beetles at control bait/non-bait area minus total number of beetles at infected
bait/non-bait area such that a positive value indicates preference for uninfective feces.
2n = 12, n = 12, d.f. = 22 in all cases; data from Tables 2 and 3.
Table 2. Total number of beetles occurring at each bait type (infected or control) summed over the entire 60 minute trial (range in
parenthesis) with mean number of beetles (standard errors in parenthesis).
Males Females
Trial Infected Control Infected Control
1. 34 (0-3) 299 (0-8) 26 (0-4) 22 (0-1)
2. 3 (0-1) 0 (0-0) 54 (0-3) 12 (0-2)
3. 36 (0-3) 243 (0-7) 22 (0-3) 45 (0-3)
4. 33 (0-3) 198 (0-7) 24 (0-3) 161 (0-8)
5. 73 (0-7) 173 (0-7) 36 (0-2) 71 (0-5)
6. 42 (0-4) 245 (0-8) 18 (0-2) 38 (0-2)
7. 192 (0-7) 34 (0-2) 9 (0-1) 90 (0-5)
8. 10 (0-2) 276 (0-8) 43 (0-3) 272 (0-9)
9. 34 (0-3) 1 (0-1) 202 (0-6) 71 (0-4)
10. 2 (0-1) 8 (0-6) 19 (0-2) 37 (0-3)
11. 141 (0-5) 182 (0-6) 20 (0-2) 36 (0-2)
12. 27 (0-2) 104 (0-6) 69 (0-8) 15 (0-2)
Mean 52.3 (16.7) 146.9 (32.6) 45.2 (16.7) 72.5 (21.7)
Table 3. Total number of beetles occurring in the non-bait area for each half of the arena (infected or control) summed over the entire
60 minute trial (range in parenthesis) with mean number of beetles (standard errors in parenthesis).
Males Females
Trial Infected Control Infected Control
1. 150 (0-8) 117 (0-5) 254 (0-10) 298 (0-9)
2. 238 (1-10) 359 (0-9) 265 (1-8) 269 (1-8)
3. 179 (0-7) 142 (0-6) 244 (0-7) 289 (2-9)
4. 163 (0-6) 206 (0-7) 208 (0-8) 207 (0-7)
5. 164 (0-8) 190 (0-7) 215 (0-8) 278 (1-8)
6. 158 (0-7) 155 (0-5) 301 (0-10) 243 (0-9)
7. 208 (0-8) 166 (0-9) 204 (0-7) 297 (2-9)
8. 136 (0-6) 178 (0-8) 124 (0-5) 161 (0-8)
9. 206 (1-6) 359 (4-8) 160 (0-7) 167 (0-6)
10. 324 (1-9) 266 (1-9) 315 (3-8) 229 (0-7)
11. 153 (0-7) 126 (0-6) 265 (0-9) 279 (0-10)
12. 217 (0-7) 252 (0-9) 253 (1-8) 263 (1-9)
Mean 191.3 (15.1) 209.6 (24) 234 (16) 248.3 (13.9)
J. F. Shea / Natural Science 2 (2010) 855-859
Copyright © 2010 SciRes. OPEN ACCESS
did not differ between males and females at the infective
(F1, 20 = 0.211, P = 0.651) or the control non-bait areas
(F1, 20 = 0.748, P = 0.397).
The presence of parasites often restricts host movement
[18,19]. In this study, male and female beetles did not
differ in the number of occurrences in the non-bait re-
gions suggesting that beetle movement is not restricted
by the presence of feces or tapeworm eggs. Thus, fewer
beetles at the infective feces suggest that male beetles
avoid infective feces rather than feces in general. It is
unlikely that the control bait repels males since the total
number of males in the non-bait regions did not differ
between infected and control halves of the arena. Also,
male and females did not differ in the maximum number
of occurrences at either the infective or control baits.
This indicates that both sexes behave similarly when
foraging in unisex groups, minimizing concern about the
confounding influence of social interactions. Overall,
these data indicate that male beetles avoid infective feces
while females show no preference.
Two experiments with T. molitor examined the costs
of infection on fertility. In the first, males mated singly
with infected females produced an average (n = 30) of
115.2 ovulated eggs, while males mated with uninfected
females produced an average (n = 30) of 127.9 ovulated
eggs [20]. In the second, females mated singly with in-
fected males produce an average (n = 7) of 37 larvae
while females mated with uninfected males produce an
average (n = 13) of 63.7 larvae [21]. Additionally, in-
fected male T. molitor are less attractive to females [21]
and exhibit a lowered response to pheromone produced
by uninfected females [22]. Thus, infection in males not
only results in less reproductive success, but infected
males are less likely to mate with a female.
Further, experimental infections of T. molitor indicate
that males are more susceptible to infection than females
[5], although this may not be true in some strains of Tri-
bolium confusum and Tribolium castaneum [23]. In ad-
dition, males are more exposed to infection when they
consume more food [24]. If males are more susceptible
and exposed to infection and incur a greater reproductive
cost once infected, then selection to avoid infective feces
may act stronger on males than on females. Females also
incur a cost when infected [20] and so may also avoid
infective feces, but this behavior was not detected in this
Beetle age may factor into the relative cost of infec-
tion. The finding that starved male beetles avoid infec-
tive feces only partially agrees with Pappas et al. who
found that fed male beetles avoid infective feces while
more fed and starved females fed upon infective feces
[5]. Pappas et al. tested 14-18 day old beetles whereas
this study tested seven to ten day old beetles. Repeating
this experiment with recently emerged beetles could
yield different results. This is because greater reproduc-
tive output occurs when female beetles are mated with
males infected two days post-emergence, suggesting an
example of fecundity compensation [25]. Further both
males and females experience increased survivorship
when infected two days post-emergence [26]. Thus, re-
cently emerged beetles may experience less selective
pressure to avoid infective feces.
Although the results suggest that males avoid infec-
tive feces, the effectiveness of this avoidance behavior in
natural settings needs examination because one study of
natural populations of Tenebrio molitor found that 51%
of males were infected as opposed to 41% of infected
females [27]. Also, beetles produce both sex and aggre-
gation pheromones [28,29], which could influence indi-
vidual foraging behavior, suggesting a need for experi-
ments that track marked individual beetles.
This study reflects work done while at The Ohio State University. I
thank Jerry Downhower, Peter Pappas, Tom Waite and Larry Phelan
for their helpful comments in the preparation of this manuscript. I
especially thank Peter Pappas for the use of his laboratory equipment.
Finally, I thank Yimei He for her statistical advice.
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