Brent B. Nickol

Cryptic speciation and patterns of phenotypic variation of a highly variable acanthocephalan parasite

An investigation of a parasite species that is broadly host‐ and habitat‐specific and exhibits alternative transmission strategies was undertaken to examine intraspecific variability and if it can be attributed to cryptic speciation or environmentally induced plasticity. Specimens of an acanthocephalan parasite, Leptorhynchoides thecatus, collected throughout North America were analysed phylogenetically using sequences of the cytochrome oxidase I gene and the internal transcribed spacer region. Variation in host use, habitat use, and transmission were examined in a phylogenetic context to determine if they were more likely phylogenetically based or due to environmental influences. Results indicated that most of the variation detected can be explained by the presence of cryptic species. The majority of these species have narrow host and microhabitat specificities although one species, which also may comprise a complex of species, exhibits broad host and habitat specificity. Alternate transmission pathways only occurred in two of the cryptic species and correlate with host use patterns. Taxa that mature in piscivorous piscine hosts use a paratenic fish host to bridge the trophic gap between their amphipod intermediate host and piscivorous definitive host. One potential example of environmentally induced variation was identified in three populations of these parasites, which differ on their abilities to infect different host species.

First Sequenced Mitochondrial Genome from the Phylum Acanthocephala(Leptorhynchoides thecatus) and Its Phylogenetic Position Within Metazoa

The complete sequence of the mitochondrial genome of Leptorhynchoides thecatus (Acanthocephala) was determined, and a phylogenetic analysis was carried out to determine its placement within Metazoa. The genome is circular, 13,888 bp, and contains at least 36 of the 37 genes typically found in animal mitochondrial genomes. The genes for the large and small ribosomal RNA subunits are shorter than those of most metazoans, and the structures of most of the tRNA genes are atypical. There are two significant noncoding regions (377 and 294 bp), which are the best candidates for a control region; however, these regions do not appear similar to any of the control regions of other animals studied to date. The amino acid and nucleotide sequences of the protein coding genes of L. thecatus and 25 other metazoan taxa were used in both maximum likelihood and maximum parsimony phylogenetic analyses. Results indicate that among taxa with available mitochondrial genome sequences, Platyhelminthes is the closest relative to L. thecatus, which together are the sister taxon of Nematoda; however, long branches and/or base composition bias could be responsible for this result. The monophyly of Ecdysozoa, molting organisms, was not supported by any of the analyses. This study represents the first mitochondrial genome of an acanthocephalan to be sequenced and will allow further studies of systematics, population genetics, and genome evolution.

ACANTHOCEPHALANS FROM CRABS IN THE SOUTHEASTERN U.S., WITH THE FIRST INTERMEDIATE HOSTS KNOWN FOR ARHYTHMORHYNCHUS FRASSONI AND HEXAGLANDULA CORYNOSOMA

Cystacanths of Hexaglandula corynosoma were discovered in the fiddler crabs Uca spinicarpa and Uca rapax collected in Mississippi and Florida. Adults were present in yellow-crowned night herons, Nyctanassa violacea, collected in Florida. Cystacanths are very similar to adults, varying only in size and state of development. This is the first record of H. corynosoma from the United States and the first record of an intermediate host for any species of the genus. Cystacanths of Arhythmorhynchus frassoni were present in U. rapax from Florida. The proboscis size, shape, and armature of the cystacanths are identical to those of adults from clapper rails (Rallus longirostris) collected in several southeastern states. This is the first report of an intermediate host for A. frassoni. Profilicollis altmani cystacanths were collected from mole crabs (Emerita talpoida) at Atlantic Beach, North Carolina. These specimens, together with adults obtained from shore birds in Louisiana and Mississippi, are consistent with the view that P. altmani, Profilicollis kenti, and Profilicollis texensis are synonyms, with P. altmani having nomenclatural priority.

DEVELOPMENTAL RELATIONSHIPS BETWEEN ACANTHOCEPHALANS AND ALTERED PIGMENTATION IN FRESHWATER ISOPODS

Only small (4.0-5.0 mm) Asellus intermedius could be infected routinely with Acanthoceph- alus jacksoni in the laboratory. Acanthocephlus jacksoni required 90 days at 20 to 25 C to complete development. Thirty days after exposure to parasite eggs, infected isopods could be distinguished visually from uninfected, control isopods on the basis of altered pigmentation. Study of the development of normal integumental pigmentation of Lirceus garmani, L. lineatus, and As. intermedius showed that pigmentation becomes rapidly established during growth of isopods from approximately 2 to 5 mm in length. Suscep- tibility of isopods to infection at this critical time during ommochrome pigment production, and the (statistical) relationship of parasite growth to isopod growth during the 90 days of acanthocephalan de- velopment, support the hypothesis that North American species of Acanthocephalus interfere with the metabolic pathway for ommochrome pigmentation of isopod hosts.

SITE SELECTION, GROWTH, AND SURVIVAL OF LEPTORHYNCHOIDES THECATUS (ACANTHOCEPHALA) DURING THE PREPATENT PERIOD IN LEPOMIS CYANELLUS

Lepomis cyanellus were fed 10, 20, or 40 Leptorhynchoides thecatus cystacanths, and examined after 1, 3, 7, 14, 28, or 56 days. Some cystacanths remained in the stomach 1 day, and in the small intestine for 7 days. By 14 days, worms were restricted to the pyloric ceca and mesenteries. Parenteric L. thecatus, first observed at 14 days, were encapsulated and destroyed by host cells. After 8 wk, cecal males averaged 3.7 mm in length, and females, 4.8 mm. The number of worms recovered from fish fed 40 cystacanths declined between days 3 and 14, to levels similar to those observed in fish fed 20 cystacanths. No decline was detected at either of the lower dosages. Leptorhynchoides thecatus establishes and matures in the pyloric ceca of this host species. Parasites that enter the mesenteries must be capable of surviving in the alimentary canal for at least 7 days. Lepomis cyanellus is a poor transport host for L. thecatus. Parasite activation and site selection are density-independent processes, but establishment and survival are apparently density-dependent. Lepomis cyanellus provides sufficient resources for the establishment of 10 to 15 L. thecatus. Excess parasites are lost within 14 days. Surveys indicate that Leptorhynchoides thecatus is a common, widely distributed parasite of centrarchid fishes in North America (Lincicome and Van Cleave, 1949; Lang and Edson, 1976; Samuel et al., 1976). However, DeGiustis (1949) experimental infections of Ambloplites rupestris, performed to confirm the life cycle, are the sole source of information regarding growth, maturation, site selection, and parenteric migration. Each infection was initiated with a fixed, relatively small (10) number of cystacanths, and thus little information on parasite survival and population processes was provided. Weekly recruitment of L. thecatus in Lepomis macrochirus tethered in Gull Lake, Michigan, has been studied (Esch et al., 1976), but the results are puzzling. Although pyloric ceca are considered the primary infection site (DeGiusti, 1949; Venard and Warfel, 1953), Esch et al. (1976) found only 25% of newly recruited L. thecatus in the ceca. Tethered fish recruited L. thecatus throughout the summer, but an accumulation of parasites in untethered fish from the vicinity of the cages was not documented. In the absence of concomitant loss, accumulation is a necessary consequence of a continuous recruitment process. Received 6 April 1981; revised 29 June 1981, 20 October 1981; accepted 9 December 1981. * Present address: 15 Ridgeland Circle, Wallingford, Connecticut 06492. Clearly, there are considerable gaps in our knowledge of this ubiquitous parasite of fishes. Our study, designed to examine population processes in Lepomis cyanellus, an important host in northern Nebraska (Samuel et al., 1976), provides new information on growth, maturation, site selection, and parenteric infections. Many conclusions DeGiusti (1949) derived from A. rupestris cannot be extended to L. cyanellus. MATERIALS AND METHODS Leptorhynchoides thecatus were obtained from naturally infected fish seined at Rice Creek, Oswego Co., New York. Eggs were refrigerated at 4 C within female worms until used to infect amphipods. Hyalella azteca, obtained from Lancaster Co., Nebraska, were exposed to eggs for 1 day in small, aerated plastic cups. The entire contents of the cup were then poured into 9 x 19 x 30-cm plastic culture boxes containing water and algae. In order to allow for growth and development of cystacanths, amphipods were maintained in aerated culture boxes for at least 35 days, and fed spinach. Cystacanths were dissected from amphipods and held up to 1 hr in 0.25% saline before being fed to fish. Several batches of amphipods were required to obtain sufficient parasites for this study. The relative infectivity of parasites from certain batches was quite low, and fish fed less infective cystacanths were excluded from portions of the analysis. Lepomis cyanellus were obtained in Lancaster Co., Nebraska, where Leptorhynchoides thecatus does not occur (Samuel et al., 1976). Fish were isolated in 4-liter glass jars, and fed canned Ken-L-Ration dog food at 2or 3-day intervals. Prior to the experimental period, fish were trained to accept foodfilled No. 5 gelatin capsules. Any fish larger than 55

Effect of cystacanth body size on adult success.

Laboratory-reared cystacanths of Leptorhynchoides thecatus (Acanthocephala: Rhadinorhynchidae) were used to study the effect of cystacanth size on adult success and the factors that influence cystacanth size within the intermediate host. To assess how host size and intensity of infection influence cystacanth size, infected amphipods (Hyalella azteca) were measured, and sex, length, and width of cystacanths were determined. After a subset of cystacanths was measured, small- and large-size classes of cystacanths were designated. To determine how cystacanth size relates to adult size, green sunfish (Lepomis cyanellus) were fed 10 large or small cystacanths. Fish were dissected 6 wk after infection, and worms were removed. After adult worms were permanently mounted on slides, their length and width were measured. Intensity of infection and amphipod size significantly influenced cystacanth size in that large amphipods harbored larger cystacanths than did small amphipods and heavy infections produced smaller cystacanths than did light infections. Adult worms from the small and large cystacanth–size classes showed no significant difference in size; however, large cystacanths had a significantly higher establishment and survival than did small cystacanths: 40% of large worms and 14% of small worms were recovered. The results of this study indicate that host size and host sharing influence cystacanth size and that cystacanth size is an important factor in determining adult success.

Reintroduction of Profilicollis Meyer, 1931, as a genus in acenthocephala: significance of the intermediate host.

Polymorphid acanthocephalans with long necks, spheroid proboscides, and eggs without polar swellings of the fertilization membrane constitute a morphologically distinct group of species. In the past, this taxon was considered a separate genus, Profilicollis. More recently, workers have been unwilling to accept these features as generic-level characters, and presently the group is considered a subgenus of Polymorphus. An analysis prompted by our studies of various polymorphid cystacanths in crabs along the coasts of Scotland and the U.S.A. reveals that all records of Polymorphus in decapods refer to species with 6 cement glands frequently assigned to Hexaglandula, to incidental occurrences of species purported to be Polymorphus minutus, or to species of the subgenus Profilicollis. Occurrence in decapod crustaceans implies substantial life history differences from the other species of the genus occurring in amphipod crustaceans. We conclude this, together with morphological distinctiveness, justifies return of Profilicollis to full generic status within Polymorphidae.

The Genus Centrorhynchus (Acanthocephala) in North America with Description of Centrorhynchus robustus n. sp., Redescription of Centrorhynchus conspectus, and a Key to Species

Reexamination of the type series of Centrorhynchus wardae Holloway, 1958, and Centrorhynchus conspectus Van Cleave and Pratt, 1940, revealed no consistent difference between the 2 purported species; therefore, C. wardae is considered a junior synonym of C. conspectus. Specimens from additional collections are consistent with this view and provided adequate material for redescription of the species. Centrorhynchus robustus n. sp. resembles C. conspectus in the number of hooks per Salient differences between the 2 species are size and body shape, with C. robustus having a robust trunk with the widest diameter anteriad and C. conspectus being elongate with no region of the trunk consistently widest. Additionally, a conspicuous posteriad swelling exhibited by females of C. conspectus is lacking in C. robustus. Centrorhynchus kuntzi is reported for the first time from Montana and Nebraska, and C. conspectus is reported for the first time from an eastern screech owl, Otus asio.

Copulation and sexual congress of Leptorhynchoides thecatus (Acanthocephala).

Pairs of the acanthocephalan Leptorhynchoides thecatus from laboratory infections were observed copulating at 21 days, 5 wk, and 12 wk postinfection (PI) in green sunfish, Lepomis cyanellus. Additionally, copulating pairs of worms from natural infections were observed. In each instance of paired males and females, the male was in 1 pyloric cecum of the fish and the female in another. Each had its posterior end protruding into the intestinal lumen at the point from which the ceca arise. The completely extruded bursa of the male fully enclosed the posterior end of the female in a firm attachment. In 1 instance at 5 wk PI, 2 males were observed in copula. One male had its copulatory bursa completely retracted, and the copulatory bursa of the other male was positioned just as though the mate were a female. Based on these and previous findings, it is concluded that male acanthocephalans mate indiscriminately and often throughout their lives. Habitation of ceca maximizes sexual congress because individuals often are positioned with their posterior ends extending into the intestinal lumen within the small area from which ceca originate. Emigration to find a mate is unnecessary.

PARASITE POPULATION REGULATION: LETHAL AND SUBLETHAL EFFECTS OF LEPTORHYNCHOIDES THECATUS (ACANTHOCEPHALA: RHADINORHYNCHIDAE) ON HYALELLA AZTECA (AMPHIPODA)*

The hypothesis that Leptorhynchoides thecatus populations are regulated by mortality of heavily infected intermediate hosts, Hyalella azteca, was examined experimentally. Mortality related to the infection process could not be demonstrated because no survivorship difference occurred between amphipod groups exposed to L. thecatus and unexposed control groups after 24 hr. Likewise, amphipod mortality could not be associated with infection intensity during this period. Amphipod mortality, growth, and infection intensity were monitored for 25 days after a 24-hr exposure period. Cystacanths were infec- tive to fish after 25 days. Survivorship of exposed amphipods was at least as great as that of unexposed controls. Infection intensity in exposed male survivors did not differ significantly from that in males that died during the study. Infection intensities in females and survivors of undetermined sex were signifi- cantly higher than in Hyallela that died. The effect of time on amphipod size (as indicated by head length) was significant for male survivors and all females, but not for males that died. No negative effect of infection intensity on growth was demonstrated, nor was there significant correlation between amphipod size and infection intensity for either sex of amphipod at any time during the 25-day experiment. No evidence of lethal effects or sublethal effects on growth was detected.