Rhadinorhynchus oligospinosus n. sp. (Acanthocephala, Rhadinorhynchidae) from mackerels in the Pacific Ocean off Peru and related rhadinorhynchids in the Pacific, with notes on metal analysis

Specimens of a new species of Rhadinorhynchus Lühe, 1911 are described from the chub mackerel Scomber japonicus (Scombridae) and the Chilean Jack mackerel Trachurus murphyi (Carangidae) (possibly a subspecies of Trachurus symmetricus) from the Pacific Ocean off the Peruvian coast at the Port of Chicama, La Libertad. Specimens of Rhadinorhynchus oligospinosus n. sp. are somewhat small having 11–14 rows of alternating proboscis hooks with 20–22 hooks each with posteriormost hooks in a continuous ring. Ventral hooks are robust with prominent roots but dorsal hooks are slender and shorter with discoid roots. Trunk spines are in two zones separated by a non-spiny region. Anterior trunk spines are in 2–3 complete circles but posterior spines are only ventral and lateral, and do not extend posterior to the level of the posterior end of the proboscis receptacle in both sexes. The new species is closest to Rhadinorhynchus seriolae (Yamaguti, 1963) Golvan, 1969 found in Japanese and Australian waters, but not as close to 19 other species found in the same Pacific waters off Australia, Japan, and Vietnam. In R. seriolae, posterior trunk spines extend well past the receptacle in females, among other diagnostic differences. Proboscis hooks of the new species were analyzed for chemical elements using X-ray in conjunction with EDAX (energy-dispersive analysis for X-ray) software; sulfur had a higher concentration at the edge than the middle of cut hooks.


Introduction
An increasing number of research publications on the acanthocephalan parasites of marine fishes in the east Pacific Ocean off the coast of Peru have made their appearance in the past few years. For example, Oliva et al. [29] reported on the ecology of metazoan parasites of Stellifer minor (Tschudi), Tantaleán et al. [38] surveyed the acanthocephalans of Peru, and Amin et al. [3] redescribed Rhadinorhynchus ornatus Van Cleave, 1918 from skipjack tuna, Katsuwonus pelamis (Linn.) with special reference to the presence of microtriches on its trunk, Cruces et al. [8] reported on the parasites of the chub mackerel, Scomber japonicus Houttuyn off Peru, and Minaya et al. [24] discussed the community of parasites of the Peruvian weakfish, Cynoscion analis (Jenyns) from the Peruvian Pacific. Additional reports of parasites of S. japonicus from the Pacific Ocean off the central zone of Peru include those of Luque and Iannacone [23], Rivera et al. [32], Iannacone and Luque [17], Llerena et al. [22], Oliva et al. [29], Cabrera and Tantaleán [6], and Ruelas and Cordova [33][34][35].
Elsewhere in Pacific South America, Oliva et al. [30] studied the metazoan parasites of S. japonicus of the Atlantic Ocean (Brazil) and the Pacific Ocean (Chile), and Munoz and Olmos [25,26] revised the parasitic fauna of S. japonicus from Chile. The parasitic fauna of Trachurus murphyi Nichols or Trachurus symmetricus murphyi Nichols is rarely reported; see Oliva [27,28] who reported no Rhadinorhynchus in Peruvian or Chilean waters and George-Nascimento [12] who reported Rhadinorhynchus trachuri Harada, 1935 off Chile.
Our studies from S. japonicus and the Chilean Jack Mackerel Trachurus murphyi Nichols in the Peruvian Pacific revealed the presence of a new species of Rhadinorhynchus. The latter fish species is considered a subspecies of Trachurus symmetricus Ayres by many [9,10,31]. Specimens of the same species have been misidentified as Rhadinorhynchus pristis (Rudolphi, 1802) in other reports from Peru in other collections [8]. ''Rhadinorhynchus pristis'' was not found in S. japonicus from Callao, Peru or from Antofagasta, Chile but was reported from S. japonicus in Rio de Janeiro, Brazil and Madeira, Portugal [30]. Our description adds a new taxon to the acanthocephalan fauna of the Pacific Ocean in South America. Re-examination of the Acanthocephala of S. japonicus and T. murphyi with the aim of correcting previous records would be highly desirable.

Collections
Thirty-one specimens of S. japonicus were obtained from the local fish market of the Port of Chicama, Ascope Province, La Libertad, Peru (07°42 0 S, 79°37 0 W) in March, 2014. The specimens were immediately fixed in 5% formalin and transferred to the Faculty of Biological Sciences, Ricardo Palma University, Santiago de Surco, Lima, Peru for dissection and recovery of parasites. In all, 12 male and 19 female hosts measured 30.7-38.6 cm (mean of 34.2 cm) and weighed 487-516 g (495 g). Findings on 12 species of external and internal helminth parasites, including a species of Rhadinorhynchus conspecific with our new species misidentified as R. pristis, were also reported from the same collection by Cruces et al. [8]. Additional specimens of the same species of Rhadinorhynchus were also collected from S. japonicus and from the Chilean Jack Mackerel Trachurus murphyi by Biologa Asucena Naupay, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos. They were initially fixed in 70% ethanol and 5% formalin and made available to us. A total of 21 specimens were transferred to our Arizona, USA laboratory for processing and further studies. Six specimens were used for scanning electron microscopy (SEM) studies, 3 specimens were saved for future DNA analysis, and 12 specimens (8 males, 4 females) were processed for optical microscopy as follows.

Study of Acanthocephala
Worms were punctured with a fine needle and subsequently stained in Mayer's acid carmine, destained in 4% hydrochloric acid in 70% ethanol, dehydrated in ascending concentrations of ethanol (24 h each), and cleared in 100% xylene then in 50% Canada balsam and 50% xylene (24 h each). Whole worms were then mounted in Canada balsam. Measurements are in micrometers, unless otherwise noted; the range is followed by the mean values between parentheses. Width measurements represent maximum width. Trunk length does not include proboscis, neck, or bursa. Line drawings were created by using a Ken-A-Vision micro-projector (Ward's Biological Supply Co., Rochester, NY), which uses cool quartz iodine 150 W illumination. Color-coded objectives, 10·, 20·, and 43· lenses, are used. Images of stained whole mounted specimens were projected vertically on 300 series Bristol draft paper (Strathmore, Westfield, MA), then traced and inked with India ink. Projected images were identical to the actual specimens being projected. The completed line drawings were subsequently scanned at 600 pixels on a USB and then downloaded to a computer.
Type specimens were deposited in the University of Nebraska's State Museum's Harold W. Manter Laboratory of Parasitology (HWML) collection in Lincoln, NE, USA.

SEM (scanning electron microscopy)
Samples of parasite that had been fixed and stored in 70% ethanol were processed following standard methods [21]. These included critical point drying (CPD) in sample baskets and mounting on SEM sample mounts (stubs) using conductive double-sided carbon tape. Samples were coated with gold and palladium for 3 min using a Polaron #3500 sputter coater (Quorum (Q150 TES) www.quorumtech.com) establishing an approximate thickness of 20 nm. Samples were placed and observed in an FEI Helios 660 NanoLab DualBeam (FEI, Hillsboro, OR) scanning electron microscope, with digital images obtained in the Nanolab software system (FEI, Hillsboro, OR), and then transferred to a USB for future reference. Images were taken at various magnifications. Samples were received under low vacuum conditions using 10 KV, spot size 2, 0.7 Torr using a GSE detector.
X-ray microanalysis (XEDs), EDAX (energy-dispersive analysis for X-ray) Standard methods were used for preparation, similar to the SEM procedure. Specimens were examined and positioned with the above SEM instrument, which was equipped with a Phoenix energy-dispersive X-ray analyzer (FEI, Hillsboro, OR). X-ray spot analysis and live scan analysis were performed at 16 KV with a spot size of 5, and results were recorded on charts and stored with digital imaging software attached to a computer. The TEAM *(Texture and Elemental Analytical Microscopy) software system (FEI, Hillsboro, OR) was used. Data were stored on a USB for future analysis. The data included weight, percent, and atom percent of the detected elements, following correction factors.

Ion sectioning of hooks
A dual-beam SEM with a gallium (Ga) ion source (GIS) was used for the LIMS (liquid ion metal source) part of the process. The hooks of the acanthocephalans were sectioned using a probe current between 0.2 nA and 2.1 nA according to the rate at which the area is cut. The time of cutting was based on the nature and sensitivity of the tissue. Following the initial cut, the sample also went through a milling process to obtain a smooth surface. The cut was then analyzed for chemical ions with an electron beam (Tungsten) to obtain an X-ray spectrum. Results were stored with the attached imaging software then archived for future use. The intensity of the GIS was variable due to the nature of the material being cut.

Collections
Twenty-one specimens of a new species of Rhadinorhynchus Lühe, 1911 are described from two species of perciform fish, the chub mackerel S. japonicus (Scombridae) and the Chilean Jack mackerel T. murphyi (Carangidae). Scomber japonicus is widespread in the Indo-Pacific and is usually found in the north-western and eastern Pacific within 37 km off the coast in waters between 10 and 22°C [7]. Trachurus murphyi has been identified in the South Pacific off the coasts of Chile, Ecuador, and Peru, and around New Zealand and South Australia [1,7,39]. It is considered the southern sister species of the Pacific Jack Mackerel Trachurus symmetricus Ayres, which is usually found in pelagic and off-shore environments off the Pacific coast of North America but may extend as far south as the Galapagos Islands where it could become sympatric with T. murphyi [8]. Trachurus murphyi was considered a subspecies of T. symmetricus until Poulin et al. [31] resolved its independent specific status even though [9] and other authors still recognize it as Trachurus symmetricus murphyi Nichols. These mackerels feed on copepods and other crustacean-like mysids and euphausiids, as well as on small fish and squids but on zooplankton as juveniles [6,9,20]. None of these food items has been recognized to date as an intermediate host for rhadinorhynchid acanthocephalans in the Pacific. Etymology  slender, smaller, hooks with discoid roots (Figs. 1D, 1E). Hooks broadest basally (Fig. 2D) with prominent cortical layer and separated core in cross-sections (Fig. 2E). Neck prominent. Proboscis receptacle markedly longer than proboscis with cephalic ganglion near its middle. Lemnisci digitiform, equal, invariably a little shorter than receptacle. Gonopore terminal in males, subterminal in females, distinctly anterior to bluntly pointed posterior end (Figs. 1A, 1B, 1C).

Remarks
The specimens of R. oligospinosus described in this study are conspecific with those reported in the Peruvian Pacific from S. japonicus as Rhadinorhynchus pristis Lühe, 1911 by Cruces et al. [8]. This report addressed only prevalence parameters with no taxonomic or descriptive information except for a few SEM images of proboscis and anterior trunk. The SEM images were not of diagnostic quality yet compatible with our observations. Cruces et al. [8] diagnosis confused these specimens with those of R. pristis that are, however, large worms (females up to 70.0 mm long) with 14-16 rows of 26 hooks each, weak anterior ventral hooks, only six posterior   Golvan, 1969. 9. Rhadinorhynchus pristis sensu Solonchenko, 1968 and Kovaleva, 1970 is another species [14].  Comparisons Specimens of R. oligospinosus n. sp. are closest to those of Rhadinorhynchus seriolae (Yamaguti, 1963 Nipporhynchus seriolae Yamaguti, 1963; Rhadinorhynchus pristis sensu Fukui and Morisita, 1937] first described from yellowtail (Japanese amberjack), Seriola quinqueradiata Temminck and Schlegel (Carangidae), in the prefecture of Mie, Japan [4,36]. The two species, are, however, separated as follows. In R. seriolae, (1) the females are considerably longer (15.0-30.0 mm long) than females of R. oligospinosus (8.0-11.9 mm), (2) the proboscis has 12-13 rows of more hooks (23)(24)(25) per row each compared to 20-22 hooks per row in our specimens, (3) more ventral posterior trunk spines (11 in males, 20 in females) compared to 3-7 and 10-15 in R. oligospinosus, (4) the posterior trunk spines extended well beyond the level of the posterior end of the proboscis receptacle in female specimens of R. seriolae but not in R. oligospinosus, (5) the lemnisci are markedly shorter than the receptacle in our specimens but slightly longer in Smales's [36] specimens of R. seriolae, (6) in R. seriolae, the testes are in the mid-third of the trunk but at the posterior end in R. oligospinosus, (7) the elaborate organization of the bursa and the extensive presence of sensory structures in our specimens have not been reported in R. seriolae or any other species of Rhadinorhynchus by other observers including Fukui and Morisita [11] or Golvan [13]. Kanda's [19] Rhadinorhynchus japonicus Fujita, 1920 interpreted by Ichihara et al. [18] as R. seriolae and Ichihara et al.'s [18] own ''R. seriolae'' are of questionable identity as their proboscis had 16-18 rows of 20-26 hooks each. They [18] interpreted forms with such differences in hook counts, among other features, as ''varieties'' of R. seriolae. The validity of R. japonicus has, however, been established [4]. (8) R. seriolae and R. oligospinosus n. sp. have been reported from mackerels (Carangidae and Scombridae). The first species was found in Japan [11,18,40] and east Australia [36] but our new species is reported in the Peruvian Pacific (this paper).

X-ray scans
The X-ray scans (EDAX) of gallium cut hooks are listed in Table 1 and Figures 5 and 6. There is a high content of phosphorus and calcium in the middle and edge scans with a varying amount of sulfur. Other common elements of living organisms are also recorded (carbon and oxygen) as well as elements used for specimen preparation (gallium, palladium, gold). Sulfur has a differential higher concentration at the edge (6.91 wt.%) than the middle of cut hooks (0.82 wt.%). This element is part of the prominent outer layer of most acanthocephalan hooks and is a major contributor to the hardening process of this attachment structure. Our results are comparable to those of mammalian teeth enamel. A similar EDAX study was carried out on the proboscis hooks of Echinorhynchus baeri Kostylew, 1928 showing that the large hooks have higher calcium, phosphorus, and sulfur than miniature rootless hooks [5]. Additional studies [15,16,37] have reported on hardened hooks and teeth enamel using elemental analysis (XEDs, EDAX).

Discussion
Amin et al. [4] described two new species of Rhadinorhynchus, listed 36 others, and provided keys to all 38 valid species. Only four more species of Rhadinorhynchus were described since, from marine fishes off Australia [36]. The 42 valid species of Rhadinorhynchus and Rhadinorhynchus oligospinosus n. sp. include 20 species from the Pacific Ocean especially off Australia, Japan, and Vietnam. These species are: separated by aspinose zone, anterior trunk spines in a few complete circles, and posterior trunk spines only ventral and lateral extending posteriorly only to level posterior end of proboscis receptacle in both males and females.

Conclusions
Rhadinorhynchus oligospinosus n. sp. is the only species of the currently recognized 43 species of the genus Rhadinorhynchus that is found off the coast of Peru. Another 19 species known in the Pacific Ocean are mostly found off the coasts of Australia, Japan, and Vietnam. Most of the remaining species are found off the coasts of India, Africa, and the Arabian Sea. The closest species to R. oligospinosus is R. seriolae from Japan and Australia which is, however, distinguished from our species by the distribution of posterior trunk spines and proboscis armor, among other features. Rhadinorhynchus ornatus, a more distant species from R. oligospinosus, similarly occupies a long stretch of the Pacific off the South American coast but mostly along Colombia and Ecuador but with extended distribution from species of tuna in Atlantic North America and the Sea of Japan [3]. The key by Amin et al. [4] conveniently separates those species, and others. In X-ray scans of gallium-cut hooks, the core and edge of hooks were shown to have a high content of phosphorus and calcium but hook edge had a markedly higher concentration of sulfur. Sulfur is a major contributor of the hardening process of acanthocephalan hooks as has previously been demonstrated in other acanthocephalan species.