On Some Acanthocephalans from Wildlife in Peru, with the Designation of Neoechinorhynchus (Neoechinorhynchus) cylindratus peruensis n. subsp. and Special Notes on the Status of “Polymorphus pacificus” sensu Karl, 1967

Processing for Microscopical Examination

Creating whole mounts of adults and cystacanths varied in different labs and locations. Specimens processed in Peru including P. altmani, P. spindlatus cystacanths, P. lutzi, N. cylindratus, and I. furcatus, were stained in Borax carmine and occasionally with fast green before being dehydrated in ethanol then whole mounted. Specimens processed by us in Scottsdale, Arizona including adult P. spindlatus, and N. peruensis, were stained in Mayer’s carmine, detained in 4% hydrochloric acid in 70% ethanol, dehydrated in ethanol, cleared in 100% xylene or in graded terpineol-100% ethanol, and mounted in Canada balsam.

Processing for Histopathological Studies

Infected host tissues were fixed in buffered 10% formalin. After dehydration and embedding in paraffin, sectioned at 4-6 um, fixed on glass slides and stained in hematoxylin and eosin (H & E) or Mallory’s triple stain (MT). The slides were studied with a Zeiss compound microscope or with a BH2 light Olympus microscope (Olympus Optical Co., Osachi- Shibamiya, Okaya, Nagano, Japan).

Line Drawings

Line drawings were made by using a Ken-A-Vision micro-projector (Ward’s Biological Supply Co., Rochester, New York) which uses cool quartz iodine 150W illumination with 10X, 20X, and 43X objective lenses. Images of stained whole mounted specimens were projected vertically on 300 series Bristol draft paper (Strathmore, Westfield, Massachusetts), then traced and inked with India ink. Projected images were identical to the actual specimens being projected. Microscope images were created using 10X and 40X objective lenses of a BH2 light Olympus microscope (Olympus Optical Co., Osachi-shibamiya, Okaya, Nagano, Japan) attached to an AmScope 1000 video camera (United Scope LLC, dba AmScope, Irvine, California), linked to an ASUS laptop equipped with HDMI high-definition multimedia interface system (Taiwan-USA, Fremont, California). Images from the microscope are transferred from the laptop to a USB and stored for subsequent processing on a computer.

Optical Microscopy

Images created for this presentation were acquired using a Zeiss Axioskop Transmitted Nomarski DIC Phase Contrast Microscope Trinocular (Munich, Germany) and a Canon T3i EOS 600D DSLR Camera (Melville, New York). Measurements are in micrometers unless otherwise noted; the range is followed by the mean values between parentheses when appropriate. Width measurements represent maximum width.

Optical microscope images in quoted already published works were acquired using a BH2 light Olympus microscope (Olympus Optical Co., Osachi-shibamiya, Okaya, Nagano, Japan) attached to an AmScope 1000 video camera (United Scope LLC, dba AmScope, Irvine, California), linked to an ASUS laptop equipped with HDMI high-definition multimedia interface system (Taiwan-USA, Fremont, California). Images from the microscope are transferred from the laptop to a USB and stored for subsequent processing on a computer.

Scanning Electron Microscopy (SEM)

Specimens previously processed for SEM were fixed and stored in 70% ethanol following standard methods. These included critical point drying (CPD) (Tousimis Automandri 931.GL) and mounting on aluminum SEM sample mounts (stubs) using conductive double-sided carbon tape. Samples were sputter coated with an 80%-20% gold-palladium target for 3 minutes using a sputter coater (Quorum (Q150T ES) www.quorumtech.com) equipped with a planetary stage, depositing an approximate thickness of 20 nm. Samples were placed and observed in an FEI Helios Dual Beam Nanolab 600 (FEI, Hillsboro, Oregon) Scanning Electron Microscope (FEI, Hillsboro, Oregon). Samples were imaged using an accelerating voltage of 5 kV, and a probe current of 86 pA, at high vacuum using a SE detector.

Deposits

Specimens were deposited in the University of Nebraska’s State Museum’s Harold W. Manter Laboratory (HWML) collection, Lincoln, Nebraska or at the US National Museum (USNM) Helminthological Collection, Beltsville, Maryland. Accession numbers are listed in Table 1.

(Moniliformidae) (Figs. 1-3)

Two cystacanths on 2 separate slides labeled # 146, Rodriguez were given to us, probably in 2015 by J. Iannacone. They were collected from Rattus rattus (Linn.) in San Juan de Miraflores, Lima. The specimens (Figs. 1-3) appeared similar to the cystacanths of Moniliformis kalahariensis Meyer, 1931 examined from Meyer’s Berlin Museum für Naturkunde collection with our SEM images in Amin, et al. [9] (Figs. 8-12).

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Figures 1-3: Moniliformis monilifo_rmis from _Rattus rattus.

1. A whole cystacanth.
2. Proboscis and neck.
3. Higher magnification of the proboscis showing the shape and arrangement of hooks. Figures 4-6: Neoechinorhynchus (Neoechinorhynchus) cylindratus peruensis n. subsp. from Gambusia affinis.
4. The proboscis showing the large apical organ.
5. Posterior end of a female specimen showing the reproductive system with a near-terminal gonopore.
6. A higher magnification of the posterior portion of the female reproductive system showing the shape of the vaginal bulb, the short uterus, the para-vaginal fibrillar strands, and the near terminal position of the gonopore.

(Neoechinorhynchidae) (Figs. 5-6)

Neoechinorhynchus (Neoechinorhynchus) cylindratus peruensis n. subsp., is reported from the mosquitofish Gambusia affinis (Baird & Girard) in Pantanos de Villa. It was first described by Amin, et al. [6] with 4-line drawings and 8 light microscopy images. Amin [10] first created two subgenera in the genus Neoechinorhynchus Stiles and Hassall, 1905 with species in the subgenus Neoechinorhynchus Stiles and Hassall, 1905 being distinguished from species in the subgenus Hebesoma Van Cleave, 1928 by having eggs with concentric shells without polar prolongation of fertilization membrane. In a concept uncommon in the description of species of the Acanthocephala, the species Neoechinorhynchus cylindratus is herein divided into two subspecies. By far, the most common subspecies, neoechinorhynchus is found in marine, freshwater or estuarine fishes, amphibians and reptiles in North America and Mexico. Females are typically with subterminal gonopore at a protruding ventral posterior knob. Females of the other subgenus peruensis have a large double apical organ (Fig. 4) and a near terminal gonopore and no posterior knob (Figs. 5,6), among other differences and is restricted to a small habitat in Peru.

• Designations
• Subspecies neoechinorhynchus n. subsp. With characters of the genus and subgenus Neoechinorhynchus as per Amin [10]. Posterior end of females with sub-ventral gonopore at ventral protruding knob. Common in fish, amphibians and reptiles in North America and Mexico. Type subspecies: Neoechinorhynchus (Neoechinorhynchus) cylindratus cylindratus. The species was originally described from Micropterus salmoides (Lacépède) in Pelican Lake, Minnesota as Neorhynchus cylindratus Van Cleave, 1914 (preoccupied) then Eorhynchus cylindratus Van Cleave, 1914.

• Subspecies peruensis n. subsp. With characters of the genus and subgenus Neoechinorhynchus as per Amin [10] and characters of the species N. cylindratus as per Amin, Peña, Castro [6]. Posterior end of females with terminal to near terminal gonopore and no protruding knobs. Described from imported fish in Pantanos de Villa Wildlife Refuge, Peru. Type subspecies: Neoechinorhynchus (Neoechinorhychus) cylindratus peruensis. Type host: Gambusia affinis (Baird & Girard).

Specimens. Harold W. Manter laboratory (HWML), Lincoln, Nebraska collection no. 216402 (2 males and 2 females on 2 slides).

ZooBank LSID#F4179915-0290-427B-AA2E-FF28F8854203

• Remarks The Peruvian subspecies is further distinguished from the North American and Mexican forms by having considerably wider and more robust lemnisci, paravaginal ligaments, and duct connecting the vaginal bulb with the uterine bell glands (selective apparatus) [6]. The terminal position of the gonopore is a relic of the juvenile developmental stage before the differential development of the dorsal vs. the ventral aspects of the body wall prompting the displacement of the gonopore to a ventral position in the adults which appears to be a function of diet that have become a fixed adult trait in the polluted and degraded environment of the Lima’s Pantanos de Villa Swamp. See Amin, et al. [6] for a detailed discussion.

(Echinorhynchidae) (Figs. 7, 8)

Amin and Heckmann [5] described this acanthocephalan from Peru for the first time based on Iannacone’s donation in 2009 of one vial containing 70% ethanol-preserved specimens used for SEM studies and 7 carmine stained whole mounted specimens on 7 slides collected from the Peru coast toad Chaunus limensis. Specimens were collected in the Lima area, in July, 1987 and January, 1988. Our description included 11 SEM images showing the thick trunk, anterior trunk collar, thin anterior hooks, micropores on hooks and elsewhere in variable diameter and density, sub-ventral female gonopore, and fibrillar coat of the eggs. Pseudoacanthocephalus lutzi was reported but not described from other hosts in different locations in Peru by other observers; see Tantaleán, et al. [11]. Our SEM images [5] of P. lutzi provided good depictions of the external morphology but additional perspectives of internal structures are now revealed by our light microscope images including detail of the proboscis and hooks (Fig. 7) and a subterminal female gonopore (Fig. 8).

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Figure 7-12: Pseudoacanthocephalus lutzi from Chaunus limensis. 7. A proboscis showing the shape and arrangement of hooks. 8. The subterminal gonopore of a female specimen. Figures 9, 10. Illiosentis furcatus from Sciaena deliciosa. 9. The reproductive system of a male specimen marked by the red-stained structures. Note the relative proportions of each. 10. The anterior part of the same specimen in Fig. 9 showing part of the long lemnisci and the unequal distribution of dorsal vs. ventral trunk spines. Figures 11, 12. A juvenile Corynosoma australe from Paralabrax humeralis. 11. A whole organism. 12. An everted proboscis. Note the shape and distribution of hooks.

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Figures 13-18: 13. A juvenile Corynosoma australe from Paralabrax humeralis. The middle section of a juvenile showing the distribution of the ventral trunk spines. Figures 14-16. Adult Polymorphus spindlatus from Nycticorax nycticorax. 14. Anterior trunk showing the receptacle, strong neck, and characteristic spindle-shaped proboscis. 15. A higher magnification of the proboscis of the same specimen in Fig. 14. 16. The posterior portion of the same male specimen showing the cement glands and body wall nuclei. Figures 17, 18. Polymorphus spindlatus cystacan_ths from _Orestias agassii. 17. A cystacanth with invaginated large receptacle. 18. A cystacanth with a normally evaginated spindle-shaped proboscis comparable to that of the adult.

(Family Illiosentidae) (Figs. 20)

Amin, et al. [12] briefly referred to the unusual finding of I. furcatus Van Cleave and Lincicome 1939 from the Lorna drum Sciaena deliciosa Tschudi in Peru for the first time. Illiosentis furcatus was originally described by Van Cleave and Lincicome [13] from the southern kingfish Menticirrhus americanus (Linn.) in the Grand Isle, Louisiana at which time they noted its distribution in the West Atlantic from Cape Cod in Massachusetts, USA to northern Argentina making our finding from Peru a unique extension of its range of distribution. The distribution of the closely related Illiosentis cetratus Van Cleave, 1945 is limited to the Pacific coast of California. The light microscope images of our two specimens of I. furcatus from Peru provided an additional insight into the internal anatomy of these acanthocephalans (Figs. 9,10) that appeared compatible with the original description including the proboscis armature formula that has been corrected in Amin, et al. [13] to 12-15 rows each with 26-33 hooks per row with the presence of genital spines. The closely related I. cetratus has 14 rows of hooks each with 18-23 hooks and genital spines are absent.

(Polymorphidae) (Figs. 11-13)

Iannacone provided 4 juvenile specimens of Corynosoma sp. collected from the Peruvian rock bass Paralabrax humeralis (Valenciennes) from the Fishing Terminal Chorrillos, Lima in 2009. Later on, Chero, et al. [14] reported it from the corvina drum Cilus gilberti (Abbot) from the same locality as Corynosoma obtuscens Lincicome, 1943 which is a junior synonym of C. australe. Tantalean, et al. [11] reported juvenile C. obtuscens from 18 paratenic hosts in Peru not including P. humeralis and C. gilberti. The most comprehensive study of adult C. australe from sea lions Zalophus californianus (Lesson) by Amin, et al. [15] included morphology including specimens from California, Mexico, Argentina, Antarctica, South Australia, and Brazil, with 32 SEM images and 4 lines drawings, Energy Dispersive X-ray Analysis, and molecular analysis using Cox 1 gene and amplified 18 S ribosomal DNA. The Cox 1 analysis revealed two haplotype networks, one in the Northern Hemisphere (USA and Mexico) and the other in the Southern Hemisphere (Argentina, Brazil, and Peru).

Light microscopy of our 4 peruvian specimens show a whole body (Fig. 11), details of proboscis and hooks (Fig. 12) the distribution of ventral trunk spines (Fig. 13).

(Polymorphidae) (Figs. 14-16 & 17-18)

Adults of P. spindlatus were collected from two black- crowned night herons Nycticorax Nycticorax (Linn.) shot at Puno Bay, Lake Titicaca in the summer of 1988 [1]. The two birds yielded hundreds of small adult specimens reaching up to 6 mm in length. The specimens were described from 52 whole-mounted worms and distinguished from closely related species especially with their unique spindle-shaped proboscis. The description included 7-line drawings (male, female, proboscis, row of hooks and spines with roots, female reproductive system, eggs), and 4 images of histopathological sections. The proboscis with receptacle, enlarged proboscis, and male reproductive system in adults are depicted in figures 14-16.

We have also studied cystacanths of P. spindlatus from paratenic hosts with the proboscis retracted and everted (Figs. 17, 18) on two separate occasions. Firstly, cystacanths were collected from the liver and intestinal serosa of 64 of 304 individuals (21%) of 4 species of killifish of the genus Orestias collected by gill nets from Lake Titicaca in two locations between 1991 and 1993 (Table 1) [2]. The description was based on 33 whole-mounted specimens with 2-line drawings of a male and female specimens and others sectioned for histopathology with 2 figures. Worms infected liver capsules in hyaline envelopes more frequently than intestinal sites. The pattern of proboscis armature and trunk spines was similar to that of the adults and was distinguished from that of related species. Cystacanths were observed to be not precocious. On another occasion, developing larval stages were obtained from the intestine of the Nile Tilapia Oreochromis niloticus Linn. In Villa Swamps and documented photographically in 8 Figures [4]. The morphology of the fully developed juveniles was comparable to that observed in the Lake Titicaca material, above. The attached light microscopy images of adults and cystacanths provided new perspectives to the line drawings and images in the original descriptions of this species.

(Polymorphidae) (Figs. 20)

Cystacanths and adults of P. altmani were comprehensively studied from the Pacific mole crab Emerita analoga (Stimpson) and the Belcher’s gull Larus belcheri (Vigors) by Amin, et al. [3]. The energy Dispersive

X-ray analysis of hooks showed unusually high sulfur and diminished phosphorous and calcium levels at hook tips, but higher levels of calcium in middle hooks. Histopathology in L. belcheri showed disruption and destruction of villi, necrotic tissue, and hemorrhaging.

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Figure 19-21: Adult Profilicollis altmani from Larus belcheri. 19. A typical proboscis with characteristic shape and armature. 20. Anterior trunk with comparative sizes of receptacle vs. lemnisci, testes interphase and unequal insertion of anterior ends of cement glands. 21. Male reproductive system of the same specimen in Fig. 20 showing the extent of the length of cement glands. Figures 22, 23. Profilicollis altmani cystacanths. 22. A male cystacanth showing the division of trunk regions, the extent of the size of the receptacle and two small testes in the middle section directly posterior to the receptacle. Testes can sometimes be in the posterior trunk. 23. The slightly ovoid proboscis and the elongated neck of the cystacanth in Fig. 22. Figure 24. A typical oblately spheroid proboscis of “Polymorphus (Profilicollis) pacificus” with fewer hooks per row from Larus pipixcan identified by our Peruvian colleagues.

Our recent light microscopy study revealed additional features of the internal anatomy of the proboscis armature (Fig. 19) and male reproductive system (Figs. 20, 21). The morphology of cystacanths (Figs. 22, 23) from various locations in Peru adds to the 34 SEM images produced by Amin, et al. [3] that were compared with those from other populations from the Atlantic and Pacific coasts of North and South America in California, North Carolina, Chile, and Uruguay.

(Polymorphidae) (Fig. 24)

Karl [9] described a new species of Profilicollis from California as Polymorphus pacificus Karl, 1967 from two California shore birds (ducks), the rhinoceros auklet Cerorhinca monocerata (Pallas) and the diving duck (lesser scaup) Aythya (= Marila) affinis (Eyton), unlike the gull hosts of P. altmani. Karl [8] found it in mixed infections with P. altmani in C. monocerata and with P. botulus (Van Cleave, 1916) Van Cleave 1939 [13]. Karl [8] distinguished it from populations of P. altmani and its synonyms by the large size of its trunk, oblately spheroidal proboscis (Fig. 24) with 25-30 (28) longitudinal rows of 11-12 hooks each, much- reduced trunk spines, lemnisci longer than receptacle, a pair of cement duct pouches, two independent vaginal sphincters, and larger eggs. The hosts of Karl’s [8] P. pacificus are marine ducks, unlike those of P. altmani and its synonyms.

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Figures 25-27: Neoandracantha peruensis cystacanths from Ocypod gaudichaudii. 25. The proboscis and anterior trunk section showing its shape with two bulges. Note the comparative size and length of the lemnisci vs. the receptacle. 26. The ovoid posterior trunk and the beginning of the long tail. 27. A higher magnification of the anterior trunk showing the 3 zones of trunk spines in profile.

The trunk of Karl’s [8] P. pacificus is clearly markedly larger than that of P. altmani and its synonyms but the size and oblate proboscis is similar to that of the oblately spheroidal proboscis of the P. bullocki of Mateo, et al. [17] (Table 2). Measurements of the proboscis in the first description by Perry [16] are the smallest. The comparative proboscis hook and spine formulas decidedly overlap. The duct pouches and the distinction in the vaginal sphincters have not been observed by other observers so comparisons cannot be made, and the relatively larger egg size is similar to that of P. bullocki as per Mateo, et al. [17].

Table 2: Comparative morphometrics between populations and synonymies of Profilicollis altmani (Perry, 1942) Van Cleave, 1947 (=P. texensis & P. bullocki)1 and Polymorphus (=Profilicollis) pacificus of Karl, 1967. 1. Other synonyms include P. kenti Van Cleave, 1947. 2. Polymorphus texensis has a wide range of measurements and is included to give a good measure of intraspecific variability within Profilicollis altmani. Results of Karl’s (1967) re-examination of Webster’s type material are added between parentheses. 3. Karl (1967) reported 8 species of California shore birds naturally infected with P. altmani, with Catoptrophorus semipalmatus being the most heavily infected species. His morphometrics (in this table) were based on emended description of specimens obtained from his heavily infected experimental hosts, the duckling Anas platyrhynchus Linn. Given common measurements of proboscis, hooks, receptacle, and lemnisci were the same for both sexes. 4. Range (mean). 5. Bolded figures are exceptional extremes.

Karl’s [8] P. pacificus has not been recognized since. We are concerned about this species because certain of our Peruvian collaborators labeled some of their slides of adults as Polymorphus (Profilicollis) pacificum from Larus pipixcan Wangler, from Lima in 1976. Some of the slides of cystacanths collected from Emerita analoga by the same workers were also labeled Polymorphus (Profilicollis) pacificum. We have critically examined these whole-mounted smaller adults from Peru and light microscopical images of some of their features were compared with those described by Karl [8]. We have observed some comparability especially in the proboscis shape, size and armature (fewer hooks per row) to support Karl’s [8] description that appears, however, to depict an extreme variant of P. altmani as measurements and counts of critical structures appear to merge or overlap (Table 2) but perhaps not sufficient to confer a new species status.

(Polymorphidae) (Figs. 25-27)

The cystacanths of Neoandracantha peruensis Amin & Heckmann [7] were described from the ghost crab Ocypod gaudichaudii Milne-Edwards & Lucas from the Pacific coast off Lima, Peru. The new genus Neoandracantha Amin and Heckmann [7] has two anterior swellings (Figs. 25,26) with 3 separate fields of spines on the fore-trunk swelling (Fig. 27) which distinguished it from Andracantha Schmidt, 1975 that has only two fields of anterior trunk spines. Our specimens are slender with long tubular hind trunk (Fig. 26) void of genital spines. Our original description was supplemented with 6-line drawings, 17 SEM images, and an Energy Dispersive X-ray analysis (EDXA). The EDXA showed very low sulfur levels and high phosphorous and calcium levels in proboscis hooks and the opposite pattern was observed in trunk spines [7].