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DISPERSAL IN THE ACANTHOCEPHALAN ACANTHOCEPHALUS DIRUS

Darin A. Kopp, Daniel A. Elke, Sara C. Caddigan, Amit Raj, Lizbeth Rodriguez, Mary K. Young, and Timothy C. Sparkes*Department of Biological Sciences, DePaul University, Chicago, Illinois 60614. e-mail: tsparkes@depaul.edu

DISPERSAL IN THE ACANTHOCEPHALAN ACANTHOCEPHALUS DIRUS

Darin A. Kopp, Daniel A. Elke, Sara C. Caddigan, Amit Raj, Lizbeth Rodriguez, Mary K. Young, and Timothy C. Sparkes*Department of Biological Sciences, DePaul University, Chicago, Illinois 60614. e-mail: tsparkes@depaul.edu

ABSTRACT: In acanthocephalans, dispersal typically occurs when eggs that have been released in the intestines of definitive hosts areexpelled with the feces. We examined whether the acanthocephalan Acanthocephalus dirus adopts a strategy of dispersal in which eggsare carried into the environment by gravid females. Using a combination of field surveys and lab-based experiments, we showed thatthe A. dirus female retained eggs as they passed out of the intestines and that these eggs could develop in intermediate hosts (sediment-dwelling isopods). Lab-based behavioral experiments revealed that the bodies of gravid females were attractive to foraging isopods. Wepropose that a strategy of egg dispersal could occur in A. dirus in which eggs are carried into the environment by females. This strategycould increase transmission success by dispersing eggs closer to the sediment, rather than in the water column, and by directing thefeeding behavior of target hosts.

Dispersal strategies of helminths can be relatively diverse, with

factors such as the properties of the habitat and the feeding

behavior of hosts influencing the optimal strategy (Combes et al.,

1994; Bush et al., 2001; Moore, 2002). Contrary to this general

pattern, acanthocephalans appear to have a relatively conserved

strategy of dispersal (Kennedy, 2006). Typically, eggs are released

into the intestines of definitive hosts and dispersed with the feces.

Transmission to the intermediate hosts then occurs when these

eggs are consumed (Kennedy, 2006). Although this strategy is

associated with many acanthocephalans, some authors have

suggested that some species may adopt an alternative strategy

of dispersal in which eggs are carried into the environment by

gravid females, i.e., Polymorphus minutus, Polymorphus marilis,

and Acanthocephalus jacksoni, (Nicholas and Hynes, 1958;

Denny, 1968; Muzzall and Rabalais, 1975).

Here, we have examined the potential occurrence of this

strategy in the acanthocephalan Acanthocephalus dirus (5 A.

jacksoni 5 Acanthocephalus parksidei). Populations of A. dirus are

common in streams throughout the midwestern United States

(Amin, 1985; Crompton and Nickol, 1985), where they infect

sediment-dwelling isopods (Caecidotea spp., Lirceus spp.) as

intermediate hosts and creek chub (Semotilus atromaculatus)

as definitive hosts (Seidenberg, 1973; Muzzall and Rabalais 1975;

Camp and Huizinga, 1980; Amin, 1985; Sparkes et al., 2004).

Development in A. dirus is relatively synchronous, with im-

mature acanthellae dominating infections of intermediate hosts

during the summer and mature cystacanths dominating in-

fections of definitive hosts during late spring–early summer

(Seidenberg, 1973; Amin, 1980; Camp and Huizinga, 1980). Sim-

ilar patterns of development have been identified in European

acanthocephalans (Brattey, 1988). Inside the creek chub, A. dirus

females produce eggs which are dispersed in the stream during the

spring and summer (Seidenberg, 1973; Camp and Huizinga, 1980).

Muzzall and Rabalais (1975) proposed that eggs are carried into the

stream by female A. dirus because eggs were not present in the feces

and females were expelled from definitive hosts intact. We tested

this hypothesis using a combination of field surveys and lab-based

experiments. Field surveys were used to determine whether eggs

were released into the intestines of definitive hosts or retained by

females; lab-based experiments were used to determine whether eggs

contained in expelled females could develop in intermediate hosts.

We then used a lab-based experiment to examine whether the bodies

of gravid females were attractive to target hosts and, hence, could

facilitate transmission.

MATERIALS AND METHODS

Site of study

All organisms were collected from Buffalo Creek, located 60 kmnorthwest of Chicago in Lake County, Illinois. At this site, juvenileisopods (Caecidotea intermedius) are infected by A. dirus eggs during thesummer and creek chub (S. atromaculatus) are infected by A. diruscystacanths during the autumn, winter, and spring (Sparkes et al., 2004).Gravid female A. dirus are present in the creek chub during late spring andearly summer.

Intestinal contents of S. atromaculatus

To determine whether eggs were released into the intestines of definitivehosts, we examined the intestinal contents of creek chub. Twenty-onecreek chub were collected during the spring of 2008 and 2009 using acombination of seine and hand nets (n 5 12, 22 May 2008; n 5 9, 21 May2009). Immediately following capture, each fish was killed (MS222), fixedin formalin (10%, 24 hr), and stored in ethanol (70%) prior to necropsy.The intestinal parasites present were removed and identified and thecontents of the intestines examined for eggs using a dissecting microscope.In each case, the contents were washed into a Petri dish and both thetransferred contents and the remaining intestines were examined for eggs.If a fish contained a female A. dirus whose body was broken, it wasexcluded from the analysis because eggs present in the intestines may havebeen liberated during necropsy.

To determine whether eggs were present in female acanthocephalans, wedissected the females recovered from the creek chub and recorded thepresence or absence of eggs. For a sub-sample of these females (n 5 10),we estimated total egg content to allow the determination of therelationship between intestinal egg counts and female egg counts. Forthis analysis, each female was placed into a small Petri dish (35 3 10 mm),measured (length, width), cut into small sections, and the eggs and ovarianballs (if present) removed. Female size was estimated by calculating totalvolume (volume 5 [p 3 length 3 width2]/6; Dezfuli et al., 2001). To obtainegg counts, the eggs and ovarian balls recovered from each female weretransferred into a glass tube containing distilled water (13 3 100 mm; totalvolume 5 5 ml). Prior to each count, the tube was gently vortexed for15 sec and a 4-ml sub-sample was transferred to a Cell-VU (MillenniumSciences, Inc., New York, New York) slide (n 5 30). Each sub-sample wasplaced into the middle of a marked circle that had been divided into 8sections of equal size. One drop of Lugol’s solution was then added tostain the eggs. The number of eggs that contained acanthors was counted,the average number of eggs present per sub-sample calculated, and thetotal egg content per parasite estimated (based on the initial volume ofliquid). The same procedure was used to estimate the total number ofovarian balls present. We used a second sub-sample of the femalesrecovered from the creek chub (n 5 13) to estimate the level of eggmaturity. For each female, we removed a random sample of 30 eggs anddetermined egg maturity using the number of membranes present. Eggsthat possessed 4 membranes were considered mature (West, 1964). Thelength of the eggs was also measured.

Received 28 April 2011; revised 31 May 2011; accepted 13 June 2011.*To whom correspondence should be addressed.

DOI: 10.1645/GE-2750.1

J. Parasitol., 97(6), 2011, pp. 1101–1105

F American Society of Parasitologists 2011

1101

Experimental infection of C. intermedius

To determine whether eggs contained in expelled female A. dirus coulddevelop in intermediate hosts, we experimentally infected C. intermedius inthe laboratory. Juvenile C. intermedius were collected and transported tothe laboratory at DePaul University (25 June 2007). The isopods were thenexposed to either eggs recovered from a female or the bodies of gravidfemales.

To obtain the female A. dirus, creek chub were collected (8 May 2007)and transported to the laboratory (n 5 10). Each fish was housedindividually (38-L aquarium, filled with a mixture of stream water andconditioned tap water), and fed fish pellets ad libitum. A plastic gratepositioned approximately 4 cm from the bottom of the tank was used toisolate expelled parasites. The tanks were monitored daily for 23 days (8May–30 May 2007) and expelled females were removed and stored inconditioned tap water at 4 C (n 5 13, mean number of days prior toexpulsion 5 18). Five of these females were used for the experimentalinfections.

Experimental infections using free eggs were carried out between 30 Julyand 29 August 2007. Eggs used for the infections were removed from thebody of a female A. dirus and stored in a Petri dish (35 3 10 mm) inconditioned tap water at 4 C. Body size measurements were used tostandardize isopod size between infection groups (n 5 54 per group).Maple leaves were pre-soaked, cut into squares (100 3 100 mm), andplaced into individual Petri dishes (35 3 10 mm). Using a fine pin attachedto a probe, either 5 or 10 eggs were placed onto each leaf square. Anindividual isopod was then allowed to feed in a dish containing 1 leafsquare for 24 hr. Each juvenile was then transferred into a cup (80 3120 mm, 300 ml) containing leaf material that was partially filled withconditioned tap water and aerated. Isopods were monitored daily for30 days, after which the surviving isopods were preserved (70% ethanol),measured, dissected, and infection status and parasite size (length) wasrecorded. Body size measurements of the parasites were used to distinguishbetween field-based (pre-existing) and lab-based (experimental) infections.

Experimental infections with gravid females were carried out betweenJune and September 2007. Juvenile C. intermedius (n 5 1,000) werecollected (25 June 2007), transferred to the lab, and housed in 2 holdingarenas (55 3 45 3 25 cm, n 5 500 per group). Each arena contained amixture of stream and conditioned tap water, maple leaves obtained fromthe DePaul campus, and algal material obtained from the field site. Forthe experimental infections, 4 gravid female A. dirus were introduced intoan arena (8 August 2007, ‘experimental’ group). The other arena was leftundisturbed (‘control’ group). After 30 days, 50 juveniles were captured atrandom from each arena and preserved (70% ethanol). Isopods were thenmeasured, necropsied, and infection status and parasite length recorded.

To obtain measures of the typical infection dynamics in nature, weexamined juveniles collected from Buffalo Creek during the first month ofinfection (June). These juveniles were captured from vegetation and fromthe underside of rocks using hand nets and were preserved in 70% ethanol(n 5 140). In the lab, each isopod was measured (body length), dissected,and infection status and parasite size (length) recorded.

Feeding behavior of C. intermedius

To determine whether juvenile isopods were attracted to the bodies offemale A. dirus, we exposed isopods to either gravid females and leafmaterial or mature males and leaf material. Juvenile isopods were collectedfrom Buffalo Creek (25 June 2007), transported to DePaul University, andplaced into a holding arena (55 3 45 3 25 cm) containing aerated streamwater and detritus. Behavioral assays were conducted in a feeding arena

(8.5-cm diameter Petri dish) that was partially filled with conditioned tapwater. For each trial, individual isopods were placed in an arena with a leafsquare (1 3 1 cm) and a parasite (gravid female or mature male) for 20 min.The leaf and parasite were attached to mesh screens (2 3 2 cm) that wereplaced equidistant from the middle of the Petri dish. After an isopod wasreleased in the center of the dish, encounters and feeding attempts on boththe leaf material and parasite were recorded. An encounter was counted ifthe isopod made contact with the food item. A feeding attempt was countedif the head of the isopod remained in contact with the food item for morethan 15 sec. For each parasite, we collected behavioral data for 10 isopods.These data were converted to single values for each parasite by calculatingpercent response values, i.e., percent of isopods that encountered the fooditem and percent of isopods that attempted to feed on the item. To determinewhether isopods preferred to feed on the parasite or the leaf material, wecompared the feeding responses between food types using paired t-tests(paired because the isopods were exposed to the leaves and parasitessimultaneously). To determine whether isopods preferred to feed on gravidfemales or mature males, we compared the feeding responses of isopodsbetween parasite types using unpaired t-tests. Following the trials, theisopods were dissected and infection status determined. To resolve whetherbody size or prior infection status differed between groups, we comparedthese variables using an unpaired t-test (body size) and a G-test (priorinfection status). For analysis that required parametric tests, normality ofthe data was determined using Systat 10 (Systat, Chicago, Illinois).

RESULTS

Intestinal contents of S. atromaculatus

A total of 163 A. dirus individuals was recovered from the 21

creek chub collected from Buffalo Creek (prevalence 5 95%,

median intensity 5 5, range 5 1–31). Sixty-six percent of these

parasites were female (n 5 108), 93% of the females contained

eggs, and the average egg to ovarian ball ratio per female was

92:8. Estimates of fecundity on a sub-set of these individuals (n 5

10) revealed that the females contained an average of 40,000 eggs

(SE 5 6,800, mean female volume 5 2.4 mm3, SE 5 0.35). Six of

these parasites contained eggs and no ovarian balls and 4

contained a mixture of eggs and ovarian balls. For the 4 females

that contained a mixture, the average ratio of eggs to ovarian balls

was 98:2, indicating that most of the eggs that would most likely

be produced were present. Estimates of egg maturity, based on 30

eggs per female, revealed that, on average, 67% of the eggs were

mature (SE 5 6.1, n 5 13). Mature eggs were larger than

immature eggs (mature: mean length 5 114 mm, SE 5 1.8, n 5 13;

immature: mean length 5 74 mm, SE 5 1.2, n 5 13).

Seventeen of the original 21 creek chub contained female A.

dirus. Five of these fish were excluded from the analysis of

intestinal contents because the bodies of 1, or more, females were

broken during dissection. The results obtained for the remaining

12 fish are shown in Table I. Eight of these fish contained no eggs

in their intestines and 4 contained a small number of eggs (1, 2, 5,

and 13, respectively). To determine the expected number of

TABLE I. Intestinal contents of Semotilus atromaculatus and estimates of egg number and egg maturity for female Acanthocephalus dirus. Shown aremean (SE) or mode (range) values for each variable. The mean number of mature eggs per fish was calculated by multiplying the number of gravidfemales per fish, the mean number of eggs per gravid female (40,000), the mean proportion of mature eggs per female (0.67), and the mean proportion ofeggs to ovarian balls per gravid female (0.92:0.08).

Mean no. of gravid Acanthocephalus

dirus females per fish (SE)

Mean no. of mature

eggs per fish (SE)

No. of eggs in the intestines

per fish (mode, range)

Estimated % of eggs released

into intestine (range)

Mean, mode 3 75,000 0 0

SE, range 0.9 21,000 0–13 0–0.02

n 12 12 12 12

1102 THE JOURNAL OF PARASITOLOGY, VOL. 97, NO. 6, DECEMBER 2011

mature eggs present per fish, we used values obtained from the

parasites present in the focal fish (number of gravid females per

fish, proportion of eggs to ovarian balls per gravid female 5

0.92:0.08) and values obtained from the other sub-groups of

females (proportion of mature eggs per gravid female 5 0.67, egg

content per gravid female 5 40,000). Based on these values, the

estimated number of eggs present per creek chub was approxi-

mately 75,000. Given this estimate, the highest number of A. dirus

eggs found in the intestines would represent less than 0.02% of the

estimated number of mature eggs present per fish.

Experimental infection of C. intermedius

To distinguish between pre-existing and experimental infec-

tions, we examined variation in body size among the parasites

recovered (Fig. 1). Experimental larvae ranged in size from

0.1 mm to 1.0 mm, whereas pre-existing larvae ranged in size

from 1.0 mm to 4.8 mm. Both of these ranges fall into the typical

size-range of A. dirus at this site during the first month of

infection (Fig. 1).

For the isopods exposed to free eggs, 24% acquired new

infections (Table II). Prevalence of new infections was not

influenced by pre-existing infection (5 eggs: G 5 0.4, df 5 1, P .

0.05; 10 eggs: G 5 0.01, df 5 1, P . 0.05) and there was no effect

of egg density (5, 10) on establishment success (G 5 0.5, df 5 1,

P . 0.05). For the isopods exposed to gravid females, 88%

acquired new infections (Table II). For these isopods, there was

no effect of pre-existing infections on prevalence of new infections

(G 5 2.0, df 5 1, P . 0.05).

Feeding behavior of C. intermedius

The results obtained from the behavioral experiment are shown

in Figure 2. There was a significant difference in encounter rates

between leaf material and parasite within each experimental

group (gravid female, t 5 5.6, df 5 5, P 5 0.003; male, t 5 12.9,

df 5 5, P , 0.001), but there was no difference in encounter rates

with the different parasites types (t 5 1.5, df 5 10, P 5 0.2).

Isopods that encountered gravid females exhibited feeding

responses that were comparable to responses to leaf material

(t 5 0.2, df 5 5, P 5 0.8). In contrast, isopods that encountered

males fed more on the leaf material (t 5 6.2, df 5 5, P 5 0.002).

Comparison of the feeding responses between groups showed that

isopods were more likely to feed on females than on males (t 5

3.7, df 5 10, P 5 0.004). There was no difference in either body

size of isopods (t 5 0.02, df 5 118, P 5 0.9) or prevalence of pre-

existing infections (G 5 1.4, df 5 1, P . 0.05) between the

experimental groups.

DISCUSSION

In most acanthocephalans, dispersal occurs when eggs released

in the intestines of definitive hosts are expelled with the feces

(Kennedy, 2006). Contrary to this pattern, Muzzall and Rabalais

(1975) found that eggs of the acanthocephalan A. dirus were not

present in the feces and proposed that gravid females carried the

eggs into the environment prior to dispersal. Similar mechanisms

have been proposed for the aquatic acanthocephalans P. minutus

and P. marilis (Nicholas and Hynes, 1958; Denny, 1968).

Consistent with this type of strategy, we found that eggs did

not appear to be released into the intestines of definitive hosts by

female A. dirus, but instead were retained by gravid females when

they were expelled from the definitive host intact. Experimental

infections revealed that the eggs contained in these females could

FIGURE 1. Size distributions of Acanthocephalus dirus recovered fromjuvenile Caecidotea intermedius in both experimental infections andnatural infections. Isopods in the control group (A) were not exposed toA. dirus eggs. Isopods in the experimental group (B) were exposed to freeeggs or the bodies of gravid females. Parasites recovered from isopods (C)collected in June represent the natural infection levels during the firstmonth of infection. Lab-based infections were identified as parasites withbody lengths less than 1.0 mm (indicated by the dashed line in A and B).

TABLE II. Experimental infection of Caecidotea intermedius by Acanthocephalus dirus. Isopods were exposed to free eggs (n 5 5 or 10) or gravid females(n 5 4). Shown are mean (length) or median (intensity) values and sample sizes for isopods and parasites. Juvenile isopods collected in June from BuffaloCreek represent the natural infection levels during the first month of infection.

Source of infection Infection location No. isopods

Isopod length,

mm (SE) Prevalence % Intensity (range) No. parasites

Free eggs .Lab 55 3.6 (0.10) 24 1 (1–4) 21

Gravid females .Lab 50 4.3 (0.10) 88 5 (1–25) 257

Natural infection .Field 140 4.1 (0.07) 61 1 (1–16) 119

KOPP ET AL.—DISPERSAL IN A. DIRUS 1103

develop in intermediate hosts. These results are consistent with

the hypothesis that an alternative strategy of egg dispersal occurs

for A. dirus. However, given that acanthocephalan eggs are often

released into the intestines of definitive hosts in spurts (Crompton

and Whitfield, 1968), we cannot exclude the possibility that eggs

were also released with the feces.

Several factors associated with both the properties of the

stream habitat and host biology could potentially favor a strategy

of egg retention in which eggs are carried into the stream by

female A. dirus. In streams, dispersing eggs in the feces could be

problematic because acanthocephalan eggs are buoyant (e.g.,

George and Nadakal, 1973). Thus, eggs released in the water

column could be carried downstream and deposited in microhab-

itats that are not occupied by target hosts, e.g., surface algae. In

contrast, if eggs are retained by females, they could be carried into

the microhabitat of the target hosts when the females sink

through the water column. Egg dispersal would then occur within

the microhabitat of the sediment-dwelling hosts when the bodies

of the females degrade. Given that water velocity is reduced close

to the sediment in streams (Gordon et al., 1992), dispersal within

this microhabitat could be relatively localized. In addition,

because A. dirus eggs possess fibrils (West, 1964; Oetinger and

Nickol, 1974; Nikishin, 2001), eggs dispersed within the micro-

habitat could potentially retain their position by attaching to

structures on the sediment, e.g., leaf material. Thus, egg retention

by female A. dirus could facilitate transmission by increasing the

likelihood that eggs are dispersed within the microhabitat of the

target hosts.

Another factor that could favor egg retention by females is

that the bodies of gravid females attract foraging isopods. In the

lab-based behavioral experiment, the feeding response of isopods

to the bodies of gravid females was greater than the response to

males and comparable to the response to leaf material. Thus, the

presence of gravid females in the microhabitat of the target hosts

could potentially facilitate transmission by directing the feeding

behavior of the hosts. This type of host attraction is somewhat

consistent with other parasites that appear to attract hosts during

the free-living stage, e.g., cestodes and trematodes (Evans et al.,

1992; Beuret and Pearson, 1994).

Several authors have proposed that factors such as habitat

properties and host feeding behavior could play a significant role in

determining strategies of dispersal in acanthocephalans (Nickol,

1985; Nikishin, 2001; Kennedy, 2006). For example, eggs of

the acanthocephalans Pallisentis nagpurensis and Pallisentis rexis

expand and migrate into the water column where they can

potentially encounter copepod hosts (George and Nadakal, 1973;

Wongkham and Whitfield, 2004). Similarly, eggs of the acantho-

cephalan Leptorhynchoides thecatus attach to algae suspended in the

water column, where they are potentially able to encounter foraging

amphipods (Uznanski and Nickol, 1976; Barger and Nickol, 1998).

The results presented here are consistent with the interpretation that

eggs are carried into the microhabitat of target hosts by female A.

dirus. This strategy could be adaptive in response to both habitat

properties (stream current) and host biology (microhabitat use,

feeding behavior). However, future studies are required to

determine the importance of this strategy in nature.

ACKNOWLEDGMENTS

Funding was provided by the National Science Foundation, IllinoisLouis-Stokes Alliance for Minority Participation Program (L.R.,M.K.Y.), the Undergraduate Summer Research Program (D.A.K.,D.A.E.), the Undergraduate Research Assistantship Program (S.C.C.), aFaculty Research and Development Grant, the University ResearchCouncil (Competitive Research Grant, Paid Research Leave Program),and the Department of Biological Sciences at DePaul University.

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