Do male and female beetles (Tenebrio molitor) respond differently to rat feces containing eggs from the tapeworm, Hymenolepis diminuta?

doi:10.4236/ns.2010.28107

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Vol.2, No.8, 855-859 (2010) Natural Science

http://dx.doi.org/10.4236/ns.2010.28107

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; [email protected]

Received 13 March 2010; revised 19 April 2010; accepted 23 April 2010.

ABSTRACT

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

Manipulation

1. INTRODUCTION

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.

2. MATERIALS AND METHODS

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

856

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

preference.

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.

3. RESULTS

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

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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

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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).

4. DISCUSSION

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

experiment.

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.

5. AKNOWLEDGEMENTS

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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