Revision of Corynosoma australe Johnston, 1937 (Acanthocephala: Polymorphidae) from a North American population using novel SEM images, Energy Dispersive X-ray Analysis, and molecular analysis

Thirty-three sea lions, Zalophus californianus found stranded on the Pacific coast near San Francisco, California (37°46′ N; 122°25′ W) in February and March 2012, 2015 and 2016 were kept alive and subsequently died and examined for parasites in the Marine Mammal Center (TMMC), Sausalito, California (see Kuzmina et al., 2018 for details). A total of 1,201 adult specimens of C. australe were collected from one 2-year-old female sea lion, necropsied at TMMC in 2015 (Lisitsyna et al., 2018). Fifty-one specimens of this collection were whole mounted and studied by Lisitsyna et al. (2019) to verify the synonymy of C. obtuscens with C. australe. A sub-set of about 170 specimens from the initial larger collection was made available to Omar M. Amin (OMA) for our study. Of these specimens, 29 specimens were processed for microscopical studies, 12 for SEM, and 2 for molecular analysis. Twelve of the whole-mounted specimens were deposited at the Harold W. Manter Laboratory (HWML) parasitology collection, University of Nebraska State Museum, Lincoln, Nebraska. The remaining specimens are in the OMA collection.

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 hr each), and cleared in 100 % xylene then in 50 % Canada balsam and 50 % xylene (24 hr 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, N.Y.) which uses cool quartz iodine 150W illumination. Images of stained whole mounted specimens are projected vertically on 300 series Bristol draft paper (Starthmore, Westfield, Massachusetts), then traced and inked with India ink. The completed line drawings are subsequently scanned at 600 pixels on a USB and subsequently downloaded on a computer.

Voucher specimens were deposited at the University of Nebraska's State Museum Harold W. Manter Laboratory (HWML) collection in Lincoln, Nebraska, USA; No. 216825 (12 voucher specimens on 3 slides) were sectioned using the FEI Helios Dual Beam Nanolab mentioned above. A gas injection magnetron sputtering technique regulating the rate of cutting. The hooks of the acanthocephalans were centered on the SEM stage and cross-sectioned using an ion-accelerating voltage of 30 kV and a probe current of 2.7 nA following the initial cut. The sample goes through a cleaning cross-section milling process to obtain a smoother surface. The cut was analyzed with an X-ray normally at the tip, middle, and base of hooks for chemical ions with an electron beam (Tungsten) to obtain an X-ray spectrum. The intensity of the GIS was variable according to the nature of the material being cut. Results were stored with the attached imaging software and then transferred to a USB for future use.

The Helios Nanolab 600 is equipped with an EDXA (Mahwah, NJ) TEAM Pegasus system with an Octane Plus detector. The sectioned cuts were analyzed by EDXA. Spectra of selected areas were collected from the center and the edge of each cross-section. EDXA spectra were collected using an accelerating voltage of 15 kV, and a probe current of 1.4 nA. Data collected included images of the displayed spectra as well as the raw collected data. Relative elemental percentages were generated by the TEAM software.

DNA extraction from ethanol-fixed specimens of C. australe (n=2) was performed using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions. Sequences of the 18S rDNA and mitochondrial cox1 gene were obtained by sequencing PCR products. For 18S we used the primers 18SU467F and 18SL1310R (Suzuki et al., 2008) and PCR conditions according to Amin et al. (2022a). For cox1, we used the primers LCO1490 and HCO2198 (Folmer et al., 1994) and PCR conditions as described by Amin et al. (2022a). PCR products were electrophoresed and purified with the Purelink^TM PCR Purification Kit (Invitrogen). Sequencing was then performed in both directions with the same primers mentioned above using the Big Dye Terminator Cycle Sequencing Reaction Kit.

The sequences obtained were edited in BioEdit software (Hall, 1999) and aligned using ClustalW implemented in MEGA v.11 (Tamura et al., 2021). List of acanthocephalans used in the phylogenetic analyses is presented in Tables 2 and 3. The best nucleotide substitution model was determined for 18S (GTR + I + G) and Cox1 (HKY+G+I) based on Akaike Information Criterion in MEGA MEGA v.11 (Tamura et al., 2021). Phylogenetic analyses were performed using Maximum Likelihood (ML) and Bayesian inference (BI) methods. ML tree was computed in MEGA v.11 and 1000 bootstrap pseudoreplicates were estimated for nodal support. The BI analysis was performed in TOPALi software (Milne et al., 2009) using Markov chain Monte Carlo (MCMC) searches on two simultaneous runs of four chains for 1,000,000 generations and sampling every 100th generation. The first 25 % of the sampled trees were discarded as ‘burn-in’. Information on the sequences used in the phylogenetic analyses is presented in Table 2. Pairwise genetic distances (uncorrected p-distance model) were calculated in MEGA v. 11. The new sequences obtained for C. australe in the present study were deposited in GenBank.

Among the populations of C. australe, we examined Cox1 haplotype frequency using the DNAsp v5 program (Librado & Rozas, 2009) and calculated the number of haplotypes (H) and the haplotype diversity (Hd). The haplotype network was constructed using Network software 10.2.0.0 version (Bandelt et al., 1999).

Twelve specimens that had been fixed and stored in 70 % ethanol were processed for SEM following standard methods (Lee, 1992) These included critical point drying (CPD) (Tousimis Automandri 931.GL) and mounting on aluminium 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 vacummusing a SE detector.

A dual-beam SEM with gallium (Ga) ion source (GIS) is used for the LIMS (Liquid Ion Metal Source) part of the process. Hooks (Halajian et al., 2020), and (5) phylogenetic reconstruction using mt Cox1 sequences, a median-joining haplotype networks based on Cox1 sequence data of C. australe and a phylogenetic tree from Maximum likelihood and Bayesian inference analysis of the Cox1 sequences for our California material.

Of the taxonomic accounts listed in Table 1, only Smales (1986) provided a redescription of C. australe from Neophoca cinerea in South Australia that included a few qualified differences noted following. Zdzitowiecki (1984) also provided a detailed redescription of C. australe from pinnipeds which, however, included differences in the interpretation of the female reproductive system; see our Fig. 36 and corrections in Figs. 33, 34, and associated text. Lisitsyna et al. (2019) included an analysis for species of Corynosoma based on cox 1 and 18S sequences. The following description is based on 29 specimens from Zalophus californianus in California. Characters reported for the first time are bolded.

General. With characters of the genus Corynosoma Lühe, 1904 (Polymorphidae). Worms small, spindle-shaped, with main longitudinal lacunar canals lateral, lemniscal, cement gland, and hypodermal nuclei fragmented, and ligament sacs in females single, not persistent. Worms with slight sexual dimorphism in size of trunk, proboscis, hooks, and other structures in common (Figs. 1, 2). Praesoma, proboscis, and anterior receptacle bent ventrad. Swollen fore-trunk tapers gradually to more cylindrical hind-trunk. About 6 – 8 incomplete circles of ventro-lateral spines just posterior to neck (Fig. 3) preceding complete compact circles of anterior trunk spines (crown spines) extending posteriorly to area of maximum diameter of swollen fore-trunk dorsally in both sexes (Fig. 9). Ventral extension of spines between neck and the larger genital trunk spines continuous in females (Fig. 2) but invariably interrupted posteriorly in males (Fig. 1). Distribution of ventral spines from neck to genital spines (Fig. 18) not homogeneous. Ventral space between crown spines (Figs. 9, 15) and spines on posterior cylindrical part of trunk (Fig. 17) occupied by smaller, weaker, and more thinly distributed transitional spines (Fig. 16; arrow) varying significantly in their size (smaller) and metal composition (poorer) using EDXA than other spines (Table 4). See differences in anatomy, size and strength of anterior, transitional and posterior trunk spines (Figs. 22 – 24, respectively). Continuous field of ventral trunk spines in females constricts posteriorly (Figs. 18 – 19, arrows) but never interrupted. Genital spines most robust (Figs. 20 and 25). Electron-dense micropores cover cuticular surfaces of anterior proboscis, hooks, spines, and aspinose areas of trunk (Figs. 10 – 14). See higher magnification of micropores on posterior and anterior trunk spines (Figs. 14 and 21, respectively). Proboscis long, cylindrical, slightly widening posteriorly, with elevated apical disc suggestive of surface of apical organ anteriorly (Figs. 4 – 5) and 17 – 19 longitudinal alternating rows of 11 – 14 hooks each. Hooks of 3 types. First type: anterior 7 – 9 hooks almost equally long and most slender anteriorly, with posteriorly directed, simple roots shorter than blades especially apical and subapical hooks (Figs. 6, 35 AH). Second type: Posterior 2 or 3 hooks as long as anterior hooks but more robust and wider at base with posteriorly directed roots longer than blades (Figs. 7, 35 PH). Third type: posterior-most 2 – 4 hooks (Figs. 8, 35 BH) smallest, curving, with shorter but distinct roots directed anteriorly (manubria). Neck conical with paired lateral sensory pores (Fig. 3, black arrow). Proboscis receptacle double-walled with cerebral ganglion about halfway at middle. Lemnisci equal, broad, rounded to irregular, shorter than receptacle.

Figs. 9–14.

Males. Based on 16 sexually mature adults with sperm. See Lisitsyna et al. (2019) for measurements and see additional measurements listed below. Apical, middle and basal hooks 37 – 47 (43), 40 – 45 (43), and 20 – 28 (25) long, respectively. Hook roots 30 – 37 (28), 40 – 47 (43), 15 – 22 (19), in the same order. Neck 157 – 218 (197) long by 333 – 395 (363) wide. Lemnisci 464 – 724 (602) long by 214 – 412 (325) wide. Genital spine 20 – 32 (25) in 3 – 4 circles. Reproductive system in all trunk space posterior to level of mid-receptacle. Testes relatively large, rounded, usually equal, crowded diagonally or laterally. Six pyriform cement glands, 300 – 522 (419) long by 187 – 270 (220) wide, in 3 pairs contiguous with testes, with long ducts joining 2 common cement gland ducts overlapping claviform Saefftigen's pouch, all draining into terminal genital terminalia in bursa. Bursa muscular, large, longer than wide, 364 – 800 (570) long by 364 – 605 (433) wide, with slightly undulating lip (Figs. 25, 26), and with inner plump interface studded with 2 sets of sensory papillae. Central inner set of large round papillae encircled by many rings of complex, and less elevated papillae (Figs. 27, 28) at the outer edge of which flat-headed penis (Figs. 28, 29) is found.

Figs. 15–20.

Figs. 21–26.

Females. Based on 13 gravid females with many eggs. See Lisitsyna et al. (2019) for measurements and see additional measurements below. Apical, middle, and basal hooks 42 – 50 (47), 45 – 50 (48), and 22 – 31 (27) long, respectively. Hook roots 35 – 47 (41), 45 – 57 (50), 20 – 27 (24) in the same order. Neck 166 – 260 (216) long by 343 – 437 (391) wide. Genital spines 8 – 16 in 1 – 2 circles. Lemnisci obscured by eggs. Gonopore dorso-subterminal (Fig. 30) leading anteriorly to vaginal orifice, double sphincters, very long muscular uterus in 2 parts; lower and upper uterus joined by uterine bell cells (selective apparatus; SA) near its mid-point, then uterine bell adhering to ventral body wall (Figs. 33, 34). Reproductive system measuring 2.43 – 2.44 mm in two 3.45 mm long females not congested with eggs, (70 % of trunk length). Other length measurements: vagina 186 – 189, lower uterus 707 – 718, selector apparatus 177 – 225, upper uterus 1,021 – 1,040, uterine bell 300 – 312, and width measurements: selector apparatus 146 – 156, and uterine bell 208 – 215. Lower uterus inflated anteriorly irre-spective of presence or absence of eggs in it (Figs. 33, 34). Para-vaginal muscular sheet wrapping around vagina and lining posterior tip of trunk, particularly prominent ventrally. Four para-vaginal ligament cords, short and long ones each dorsally and ventrally, originating from posterior body wall and vaginal interface dorsally and ventrally and extending anteriorly. Short cords insert in body wall just anterior to posterior tubular trunk region (para-vaginal-body wall ligament cords). Long cords reaching to base and side of uterine bell (para-vaginal-uterine-bell ligament cords) (Fig. 33). Eggs elliptic, oblong, smooth, without distinguishing markings or ornamentation (Fig. 31), with marked polar prolongation of fertilization membrane and 4 membranes (Fig. 32) with markedly lower level of metals than egg center.

Figs. 27–32.

The trunk and other structures including the proboscis, proboscis hooks, and spines (Figs. 10 – 14) had apparent osmiophilic micropores of various diameters, shapes and distribution in various body parts. In this small acanthocephalan, every possible external surface is studded with micropores. In some areas, the micropores were more widely spaced compared to the usual more widely distributed micropores more often observed in other acanthocephalan species.

The relative WT% concentrations obtained by the TEAM software for hooks, spines, and eggs of C. australe from a California sea lion, Z. californianus in California are reported in Tables 4 – 5 and Figs. 37 – 39. Our EDXA results of C. australe show a center core with a high level of sulfur at the tip of the middle and posterior hook's edge (15.38 %) and cross-section (23.52 %), respectively, but considerably lower (2.96 %) at the tip of anterior hooks. The EDXA spectra of cross sections of the middle of all hooks showed extremely high concentrations of Calcium and Phosphorus characteristic of the center core of hooks. The presence of Sulfur, Calcium, and Phosphorus in the EDXA spectra obtained from the edge of the hook base cross-section is attributed to the proximity of the exterior shell to the center core. In anterior and posterior spines, the sulfur level was highest, 12.64 and 15.61, respectively, but markedly lower in the weak middle spines at 5.97 % (Table 4) while the levels of phosphorus and calcium were minimal. The egg center had a higher level of metals especially phosphorous (7.73 %) compared to the egg shell (0.91 %) (Table 4). It is worth noting that, these reported WT% numbers should not be interpreted as compositional. They are, however, indicative of general compositional differences observed between the selected areas.

Figs. 33–36.

Figs. 37–39.

In the present study, a total of four partial 18S and Cox1 sequences of C. australe were generated. On the GenBank database, most of the sequences available for C. australe are of mt gene Cox1 and only two sequences are of 18S gene. So, for a better comparative phylogeny and to provide information related to the population relationship of C. australe we have provided a detailed analysis of Cox1 gene along with 18S gene analysis. Newly generated 18S sequences of C. australe were almost identical, shows 0.02 % genetic divergence. The sequence for C. australe from USA (MK119255) infecting the same host Z. californianus exhibited genetic divergence was 0.27 %, while with another isolate from Mexico (JX442168) infected Phocarctos hookeri divergence was 1.5 %. The phylogenetic analyses inferred with maximum likelihood (ML) and Bayesian inference (BI) methods yielded similar topologies (Fig. 40). In both phylogenetic trees, C. australe was placed with the other isolates of similar species in a clade having strong bootstrap and Bayesian probability values (Fig. 40).

Fig. 40.

In the newly generated Cox1 sequences of C. australe shows only 0.04 % intraspecific genetic divergences. Our phylogenetic tree showed that the genus Corynosoma is comprised of two main subclades – the first one A comprises specimens of C. australe from different regions in North America, i.e., the USA and Mexico, and the second one B comprises Brazil, Argentina and Peru in South America available in the GenBank database. All isolates of subclades A and B were nested in a monophyletic clade as they are the same species (Fig. 41). In the present study, Cox1 sequences of C. australe showed a strong relationship with sequences of Lisitsyna et al. (2019) from California, USA (genetic divergence was 0.9 %). The phylogenetic analyses deduced with ML and BI methods produced the same topologies. Both trees placed the specimens of C. australe in a clade obtaining strong bootstrap support and Bayesian posterior probability values (Fig. 41). The genetic divergence between the present isolates and those available in GenBank from different regions ranged from 0.9 to 3.0 %. The sequence for C. australe suggests that in comparison with other isolates of C. australe it exhibited the lowest divergence level with the isolates from the USA and the highest divergence level with the isolates from Peru (3.0 %). We also agreed with Hernández-Orts et al. (2017b) that C. hannae is a sister taxon to C. australe as both species shares close clades (Fig. 41). Another clade comprises species as different lineages as C. semerme together with C. obtuscens, C. villosum plus the sequence of C. validum, C. enhydri along with C. magdaleni isolates and a separate lineage was formed by the sequences identified as Corynosoma strumosum (Fig. 41). Lisitsyna et al., 2019 mentioned in their study that the morphological study of the isolates in the publication of García-Varela et al. (2013) was not possible due to the lack of specimens and that the species was misidentified. They also made it clear in their study that the cox1 sequence (JX442192) (Fig. 41) as well as the 18S rDNA sequence (JX442169) (Fig. 40) of C. obtuscens published by García-Varela et al. (2013) show C. semerme instead of C. obtuscens. In our study, a haplotype network was constructed inferred with 46 specimens of C. australe as shown in Fig. 42. In the complete analysis, 22 haplotypes were detected (Table 3). The H1 haplotype (H1, n = 2) are the specimens collected in the present study from the Pacific coast near San Francisco, California corresponded to specimens from North America. H2 Haplotype (H2, n = 8) are shared by C. australe populations from Mexico and Sausalito, California, the USA (approximately 10 miles north of San Francisco from where we have collected our specimens). Haplotypes H3 and H4 were found in Brazil and Argentina, respectively corresponding to the population of C. australe from South America. Haplotypes H5–H8 and H10, H11, H14, and H17 were found in Mexico corresponding to the specimens from North America. Haplotypes H9, H12, H13, H15, H16 and H20 were reported as C. australe populations of Argentina. Haplotypes H18 and H19 were found from Brazil and corresponded to specimens from South America. Furthermost haplotype (H21, n=14) and haplotype H22 were found off the Peruvian coasts (Fig. 42). A separate tree was also constructed in the present analysis for the isolates of C. australe representing clusters as in congruence of the haplotype network (Fig. 43). Additionally, isolates collected in the present study fell within a strongly supported clade representing the North American isolates of C. australe (Fig. 43).

Fig. 41.

Fig. 42.

Fig. 43.

Our report provides new insights on the morphology of structures not previously reported in the many descriptive accounts of C. australe against the backdrop of works by other observers summarized in Table 1. We explore these new insights in the following points.

The controversy of the identity of C. australe vs. that of its synonym C. obtuscens boiled down to differences in the distribution of ventral trunk spines in females, being continuous in C. obtuscens and disjunct in C. australe. A summary of these controversies is presented in Table 1 which shows that this character is variable but no plausible explanation was provided to explain this variability. We provided evidence of variable constriction in the posterior field of ventral spines (Figs. 18, 19) that when extremely restricted will produce a separation in the more anterior and posterior fields. In our specimens, the ventral trunk spines were invariably continuous but the degree of posterior constriction varied.

The micropores of C. australe, like those reported from other species of the Acanthocephala, are associated with internal crypts and vary in diameter and distribution in different trunk regions corresponding with differential absorption of nutrients. In C. australe, the micropores were distributed in most body surfaces that we have observed including the proboscis, proboscis’ hooks, trunk, and trunk spines as though to maximize the absorptive function of all surfaces of their small body. We have reported micropores in a large number of acanthocephalan species (Heckmann et al., 2013) and in a few more since, and demonstrated the tunnelling from the cuticular surface into the internal crypts by TEM. Amin et al. (2009) gave a summary of the structural-functional relationship of the micropores in various acanthocephalan species including Rhadinorhynchus ornatus Van Cleave, 1918, Polymorphus minutus (Goeze, 1782) Lühe, 1911, Moniliformis moniliformis (Bremser, 1811) Travassos, 1915, Macracanthorhynchus hirudinaceus (Pallas, 1781) Travassos 1917, and Sclerocollum rubrimaris Schmidt and Paperna, 1978. Wright and Lumsden (1969) and Byram and Fisher (1973) reported that the peripheral canals of the micropores are continuous with canalicular crypts. These crypts appear to “constitute a huge increase in external surface area. implicated in nutrient up take.” Whitfield (1979) estimated a 44-fold increase at a surface density of 15 invaginations per 1 μm² of M. moniliformis tegumental surface. The micropores and the peripheral canal connections to the canaliculi of the inner layer of the tegument were demonstrated by transmission electron micrographs in Corynosoma strumosum (Rudolphi, 1802) Lühe, 1904 from the Caspian seal Pusa caspica (Gmelin, 1788) in the Caspian Sea (Figs. 19, 20 of Amin et al., 2011) and in Neoechinorhynchus personatus Tkach, Sarabeev, Shvetsova, 2014 from flathead grey mullet Mugil cephalus Linn. 1758 in Tunisia (Figs. 26, 29, 30 in Amin et al., 2020a).

Our studies of acanthocephalan worms have usually involved EDXA of FIB-sectioned hooks and spines (Heckmann, 2006, Heckmann et al., 2007, 2012; Standing & Heckmann, 2014). Our results of chemical ions of hooks, spines, and eggs of C. australe from the California sea lion, Z. californianus in California varied from those obtained by Halajian et al. (2020, Table 1, p. 129) for a different population of the same species, C. australe, from the Cape fur seal Arctocephalus pusillus pusillus off the Namibian coast, South Africa. In specimens from South Africa, one hook had markedly lower levels of Calcium (36.10 – 43.2 %) and of Sulfur (0.2 – 0.38) (Halajian et al., 2020) compared to our specimens with 32.03 – 72.06 % and 0.34 – 3.50 % Calcium, respectively reaching 23.52 % Calcium at the tip of posterior hooks (Table 5), and spines had higher levels of Sulfur of 15.35 – 29.87 % than in our California specimens with 5.97 – 15.61 % (Table 4), among differences in other elements. The eggshell of the Namibian specimens also had considerably higher levels of Sulfur and Phosphorous. Such differences are probably related to parasite population variations, host species, and geography.

Sulfur is usually seen at the outer edge of large hooks and Calcium and Phosphorus are major ions in the base and middle of hooks where tension and strength are paramount for hook function. The following results of the EDXA of the FIB-sectioned hooks (dual beam SEM) are limited to those from our California C. australe material. The edge of the hook tips and the tip x-section of C. australe showed the highest level of Sulfur (15.38, 23.52). Comparable or higher levels of Sulfur were observed in other species of acanthocephalans. For instance, Cavisoma magnum (Southwell, 1927) Van Cleave, 1931 from Mugil cephalus in the Arabian Sea, has a similar pattern but considerably higher levels of sulfur in hook tips (43.51 wt. %) and edges (27.46 wt. %) (Amin et al., 2018). Our results are comparable to those of mammalian teeth enamel. The chemical elements present in the hooks are typical for acanthocephalans (Heckmann et al., 2007, 2012).

On the other hand, all trunk spines of C. australe had the highest level of sulfur reaching 15.61 % in posterior spines, and negligible levels of calcium and phosphorous. The middle weaker transitional spines (Figs. 16, 23) had the lowest level of sulfur of only 5.97 %. These levels are characteristic of C. australe as the level of sulfur in the anterior and posterior spines of Southwellina hispida (Van Cleave, 1925) Witenberg, 1932 was only 0.98 % and 0.96 % and the levels of calcium and phosphorous was also negligible (Table 5 of Amin et al., 2022a). In cystacanths of Profilicollis altmani (Perry, 1942) Van Cleave, 1947, another marine acanthocephalan, the pattern of sulfur levels in anterior, middle, and posterior spines was comparable to that in C. australe but the % weight was considerably lower (2.70 – 3.21 %, 2.56 %, and 3.28 – 3.94 %, respectively) and the % weight of phosphorous and calcium was considerably higher (Table 5, Amin et al., 2022a). This comparison suggests that water salinity is not involved in the distribution of elements in the spines of marine species of acanthocephalans.

The acanthor of C. australe had much higher levels of metals, especially phosphorous (7.73 %) compared to the egg shells (0.91 %) (Table 4). In terrestrial acanthocephalans such as Macracanthorhychus hirudanaceus (Pallas, 1781) infecting a mammalian host, Sus scrofa Linn.,1758, WT % of all metals, especially phosphorous was considerably higher in the cortical layer (23.48 %), compared to the middle of the egg (acanthor) (2.26 %), and moderate levels of sulfur and calcium (Table 3 in Amin et al., 2021). Phosphorous is a basic element in the hardening of egg shell, especially of terrestrial acanthocephalans and may be related to the protection of eggs from desiccation. “The 4 outer eggshell layers E1–E4 surrounding the acanthor of M. hirudinaceus were shown to contain glycoproteins (Peters et al., 1991). Glycoproteins are structural molecules forming collagen and some include phosphorus in phosphoserine (an ester of serine and phosphoric acid) in the process of P-glycosylation (Murray et al., 2006) which may explain the high Phosphorous content of 23.48 % in the cortical eggshell layer only” (Amin et al., 2021). “Results of X-ray scans of eggs are available in one other terrestrial species of acanthocephalans, Centrorhynchus globocaudatus (Zeder, 1800) Lühe, 1911 (Centrorhynchidae) were obtained from Falco tinnunculus Linn., 1758, (Falconidae) and Buteo buteo Linn., 1758 (Accipitridae) in northern Italy. Eggs of C. globocaudatus had the highest levels of sulfur in the cortical (12.94 %) and core areas (8.04 %) and a high level of Phosphorus (9.01 %) in the middle (Table 6, Fig. 32 in Amin et al., 2020b).

X-ray scan analysis provides insight into the hardened components, e.g., calcium, sulfur, and phosphorus, of acanthocephalan hooks. The EDXA appears to be species-specific, as in fingerprints. For example, EDXA is shown to have significant diagnostic value in acanthocephalan systematics. For example, Moniliformis cryptosaudi Amin, Heckmann, Sharifdini, Albayati, 2019 from Iraq is morphologically identical to Moniliformis saudi Amin, Heckmann, Mohammed, Evans, 2016 from Saudi Arabia, and it was erected based primarily on its distinctly different EDXA pattern (Amin et al., 2019) as a cryptic species. Our methodology for the detection of the chemical profile of hooks in the Acanthocephala has also been used in other parasitic groups including the Monogenea (Rubtsova et al., 2018, Rubtsova & Heckmann, 2019) and Cestoda (Rubtsova & Heckmann, 2020).

The taxonomic identity of species is deep-seated at the genetic level which is expressed by the organism's morphology and biochemistry as revealed, in part, by its elemental spectra. Amin et al. (2022a,b,c,d) discussed in detail the biological significance of EDXA as a diagnostic tool exemplified by the observation that populations of an acanthocephalan species will consistently have similar EDXA spectra irrespective of host species or geography. Metal analysis of hooks has become a diagnostic standard since hooks have the highest level of elements compared to the mid- and posterior trunk regions of the acanthocephalan body (Heckmann et al., 2012). Specifically, the Sulfur content in the proboscis is paramount in the composition of disulfide bonds in the thiol groups for cysteine and cystine of the polymerized protein molecules (Stegman, 2005). The formed disulfide bonds are direct by-products of the DNA-based process of protein synthesis which makes up the identity of a biological species. Accordingly, the level of sulfur in our EDXA profiles will indicate the number of sulfur bonds that along with the levels of calcium phosphates, will characterize the elemental identity of a species based on its nuclear DNA personality. Variations in chemical compositions probably indicate differences in allele expression. The DNA-generated sulfide bonds evident in our EDXA profiles have an important role in the stability and rigid nature of the protein accounting for the high sulfur content of the proboscis (Heckmann et al., 2012). The above processes explain the observed species-specific nature of EDXA profiles noted in our many findings.

The present study contributes further continuation to the heteropolar geographical distribution for isolates of C. australe after the work of Lisitsyna et al. (2019) and García-Varela et al. (2021) having the novel information presented in the molecular study is the inclusion of cox1 sequences of C. australe from Peru. In the present study, a total of two partial 18S and Cox1 sequences were generated for C. australe. Newly generated 18S and Cox1 sequences of C. australe were very similar and show low intraspecific genetic divergence. The sequence for C. australe of Lisitsyna et al. (2019) and García-Varela et al. (2021) from the USA and Mexico revealed a robust relationship with the present study isolates showing genetic divergence ranging between 0.9 to 1.0 %. This result suggests that the above sequence from the USA and Mexico in comparison with other populations of C. australe exhibited the lowest divergence level and that they all belong to the Northern Hemisphere. Genetic divergence among the current study population with isolates from the Southern Hemisphere ranged from 1.8 to 3.0 %. The systematic position of the C. australe isolates in the phylogenetic tree strongly suggested that the isolates fit the similar evolutionary lineage and follow the same pattern in the haplotype network (Figs. 42 and 43). For that reason, molecular data used in the studies of acanthocephalans will make available valuable information about their diversity and population studies. Our study represents the new isolates clustered together, and displayed shared haplotypes, with isolates sequenced by Lisitsyna et al. (2019) and García-Varela et al. (2021). The haplotype network in the current study was constructed using Cox1 sequences of 46 specimens of C. australe from North and South American countries. The 46 sequences of C. australe were collapsed into 22 haplotypes and our haplotype network strongly comprised two clusters based in the Northern and Southern Hemispheres. The first (I) group related to the population of C. australe from North America that includes specimens from the USA and Mexico and the study of García-Varela et al. (2021) also supports the present analysis.

The second (II) group resembled populations from South America that comprises specimens from Peru, Argentina and Brazil. These data, in association with the phylogeny evidence, support the distinct population status of C. australe. Though, the distribution of specimens of C. australe population from different regions was in consensus in representing species group at the genetic level (Figs. 42 and 43). The newly generated sequences in the present study clustered within the first (I) group clade representing species of the USA and Mexico while the specimens from Peru, Argentina and Brazil appeared associated with the second (II) group clade and these relationships were also supported by the previous study of García-Varela et al. (2021).

In the present study, molecular data clearly represented the wide distribution of a single species that might be the leading step in further understanding the speciation and evolution of this species in the future.