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

O. M. Amin; A. Chaudhary; H. S. Singh; T. Kuzmina

doi:10.2478/helm-2023-0003

Figures & Tables

SEM of whole male (Fig. 1) and female (Fig. 2) specimens of Corynosoma australe from the intestines of Zalophus californianus in California. Note the continuity of the ventral field of spines from the neck to the genital area in the female which is covered with a copulation plug (Fig. 2).

SEM of male and female specimens of Corynosoma australe from the intestines of Zalophus californianus in California. 3. Praesoma of a male worm showing the conical neck and the ventral spinose part of the anterior part of the trunk just anterior to the bulbus (white arrow) and the sensory pore (black arrow). 4. The anterior proboscis of a specimen showing the organization of the longitudinal rows of large hooks and the external appearance of the apical organ (arrow). 5. Apical view of the proboscis in Fig. 4 showing the external surface of the apical organ. 6. Lateral view of apical and subapical hooks. 7. Lateral view of middle hooks. 8. Posterior-most spine-like hooks. Note their arched curvature compared to more anterior hooks.

SEM of anterior bulbus and micropores of male and female specimens of Corynosoma australe from the intestines of Zalophus californianus in California. 9. Anterio-ventral view of the proboscis and bulbus of a female specimen. 10. Micropores at the anterior end of a proboscis. 11. Micropores on an anterior proboscis hook. 12. Micropores on anterior trunk spine. 13. Micropores at anterior end of a male trunk away from spines. 14. Micropores on the base of a posterior trunk spine in a male specimen.

SEM of spines in male and female specimens of Corynosoma australe from the intestines of Zalophus californianus in California. 15. Anterior trunk spines in the bulbus area. 16. Transition area between bulbus spines (right) and posterior trunk spines. 17–19. Continuous ventral spines in one female specimen: 17. Midventral spines. 18. Posterior ventral spines. Note that the field of spines anterior to the genital spines constricts (arrow) but remains continuous with the genital spines without breaks. 19. Another, more detailed, perspective of posterior ventral spines of the same female in Fig. 17, 18 with arrow pointing to greatest constriction. Female gonopore is on the opposite, ventral, side (dorso-subterminal). 20. Posterior end of a male specimens with invaginated bursa revealing the organization of the genital spines there.

Detail of spines in specimens of Corynosoma australe from the intestines of Zalophus californianus in California using SEM and Gallium-cut sections. 21. SEM of an anterior trunk spine in the bulbus area. 22. A longitudinal Gallium-cut section of an anterior bulbus spine. Note the structure of the core element. 23. A longitudinal Gallium-cut section of a mid-trunk spine. 24. A longitudinal Gallium-cut section of a posterior spine. Note the structural differences between the outline and core of the spine types. 25. A dorso-terminal view of the posterior end of a male specimen showing the arrangement of the genital spines. Compare with Fig. 20. 26. A lateral view of a bursa showing its distal undulations and part of the genital spines (right).

External orifices of male and female reproductive systems and eggs of Corynosoma australe from the intestines of Zalophus californianus in California using SEM and Gallium-cut sections. 27–29. The male bursa. 27. Bursa with the undulating lip, penis (white arrows), central cluster of prominent sensory bulbs (black arrows), and surrounding circles of specialized sensory domes (short black arrows). 28. The central part of a bursa showing the two types of sensory structures and the penis. 29. A higher magnification of the penis and the surrounding type of sensory domes. 30. The posterior end of a female showing the dorsal gonopore (right) and the continuity of the posterior ventral spines with the genital spines. 31. A smooth egg with no external ornamentation or dentations. 32. A cross Gallium-cut section of an egg showing 3 external egg shell membranes FM: fertilization membrane, IM: inner membrane, OM: outer membrane) with the fourth tightly enveloping the acanthor (A).

Line drawings of the female reproductive system and 3 selected hooks of Corynosoma australe from the intestines of Zalophus californianus in California. Fig. 33. The whole reproductive system of a 3.2 mm. long female. Note the presence of the Selective apparatus (SA) almost half way between the upper uterus (UU) connecting with the uterine bell (UB) and the lower uterus (LU) connecting with the dorsal vagina (V). Also note the dorsal and ventral short paravaginal ligament cords inserting in the body wall (PVBWLC) and the dorsal and ventral longer paravaginal ligament cords connecting with the uterine bell (PVUBLC). The para-vaginal muscular sheet wrapping around vagina can be observed. The continuity of trunk spines on the ventral side is evident. Fig. 34. A higher magnification of the posterior half of the reproductive system of a second female. Posterior muscular wrap not shown. Fig. 35. The 3 hook types: an anterior slender subapical hook (AH) with shorter root; a robust hook at the inflated part of the posterior proboscis (PH) with slightly longer root; and a small curved basal or near-basal hook (BH) with anteriorly directed root (manubrium). Fig. 36. The “uterine bell” of Corynosoma australe after Zdzitowiecki (1984, Fig. 2e, p. 362).

Energy Dispersive X-Ray spectra of Gallium cut anterior hook tip x-section (37), middle hook tip x-section (38), and posterior hook tip x-section (39) showing levels of sulfur in calcium and phosphorous in all hooks See Table 2 for more specific figures (bolded). Inserts: SEM of Gallium cut anterior hook tip cross-section (37), SEM of Gallium cut middle hook tip cross-section (38), and SEM of Gallium cut posterior hook tip cross-section (39). Note the high levels of calcium and phosphorous in anterior and middle hooks and the high level of sulfur in posterior hooks.

Phylogenetic relationships inferred using 18S gene sequences of Corynosoma australe and other acanthocephalan species. Nodal support from the two analyses is indicated as ML/BI and indicates values of bootstrap >70%. The scale-bar indicates the expected number of substitutions per site.

Phylogenetic reconstruction using mt Cox1 sequences of Corynosoma australe and sequences of other Acanthocephala deposited in the GenBank. The numbers indicate values of bootstrap >50%. Numbers above branches indicate nodal support as maximum likelihood (ML) and posterior probabilities from BI. The scale-bar indicates the expected number of substitutions per site.

Median-joining haplotype networks based on Cox1 sequence data of Corynosoma australe. Each circle size represents the frequency of a haplotype as in the population.

Phylogenetic tree from Maximum likelihood and Bayesian inference analysis of the Cox1 sequences of Corynosoma australe population. Outgroup: Corynosoma hannae. The scale-bar indicates the expected number of substitutions per site. Abbreviations used for isolates country: U1- present study isolates from USA, U- USA, M- Mexico, B- Brazil, A- Argentina, P- Peru. The color refers to isolates obtained as in the haplotype network (Fig. 42; red: USA, blue: Mexico, orange: Argentina, magenta: Brazil, aqua blue: Peru).

Chemical composition and localization of elements in hooks of Corynosoma australe from Zalophus californianus in California_

	Anterior hooks		Middle hooks					Posterior hooks

Element^*	Tip x-section	Middle	Longitudinal section	Tip x-section	Tip edge	Middle x-section	Middle edge	Tip x-section	Middle edge
Magnesium (Mg)	1.21	0.23–1.67^**	0.02–0.07	0.63	0.62	1.61	0.66	0.02	0.78–1.52
Sodium (Na)	0.25	0.00–0.04	0.00–0.03	0.00	0.03	0.08	0.00	0.02	0.05–0.07
Phosphorous (P)	9.95	15.74–18.71	12.78–20.35	10.64	11.00	21.32	16.10	2.82	14.91–20.49
Sulfur (S)	2.96	0.09–7.10	0.34–3.50	5.50	15.38	0.03	4.34	23.52	1.18–14.09
Calcium (Ca)	18.99	30.80–34.84	32.03–72.06	19.98	19.11	42.48	34.18	3.16	27.05–40.39

Chronological taxonomic history of Corynosoma australe from marine mammals, with special reference to ventral trunk spines in females_

Author	Host	Locality	Described as	Stage	Ventral spines
Johnston (1937)	Neophoca cinerea (Péron)	South Australia	C. australe	Adults	Discontinuous
Lincicome (1943)	Zalophus californianis (Lesson)	San Diego, California	C. obtuscens	Adults	Continuous
Van Cleave (1953)	Mycteroperca pardalis Gilbert	Mazatalán, Mexico	C. obtuscens	Cystacanth	Continuous
Morini & Boero (1960)	Otaria flavescence Shaw	Argentina	C. otariae	Adults	Discontinuous
Zdzitowiecki (1984)	Hydrurga leptonyx (Blainville)	South Shetlands, Antarctica	C. australe	Adults	25% continuous
Smales (1986)	Neophoca cinerea (Péron)Arctocephalus pusillatus Schreber	South Australia	C. australe	Adults (Figs. 10,11)	Discontinuous
Zdzitowiecki (1991)	Few “suitable” definitive & paratenic hosts	Antarctica	C. australe	Adults (Figs. 15 b, e)	Discontinuous
Sardella et al. (2005)	Arctocephalus australis (Zimmerman)Mirounga leonina (Linn.)Cynoscion guatucupa (Cuvier)	Argentina	C. australeC. australeC. australe	AdultsAdultsCystacanths	85–100% continuous85–100% continuous85–89% continuous
Aznar et al. (2012)	Otaria flavescens (Shaw)	Argentina	C. australe	Adults (Fig. 1B)	Discontinuous
Hernández-Orts et al. (2017a)	Spheniscus magellanicus (Foster)	Brazil	C. australe	Adults (Fig. 2B)Adults (Fig. 4B)	Continuous & 82–89% continuous
Lisitsyna et al. (2018)	Zalophus californianus (Lesson)	SausalitoCalifornia	C. obtuscens	Adults	Continuous
Lisitsyna et al. (2019)	Zalophus californianus (Lesson)	SausalitoCalifornia	C. obtuscens & C. australe	Adults	1% discontinuous
Present paper	Zalophus californianus (Lesson)	SausalitoCalifornia	C. australe	Adults	Continuous with post. constriction

List of acanthocephalan species used for phylogenetic analysis based on the mt Cox1 gene sequences_ Newly generated sequences are presented in bold_

Species	Host	Host origin	GenBank accession nos.	References
Corynosoma australe	Zalophus californianus	USA	ON619618, ON614719	present study
	Zalophus californianus	USA	MK119245–MK119249	Lisitsyna et al., 2019
	Zalophus californianus	Mexico	MT676808–MT676818	García-Varela et al., 2020
	Merluccius hubbsi	Argentina	MT676819–MT676822	García-Varela et al., 2020
	Raneya brasiliensis	Argentina	MT676823–MT676824	García-Varela et al., 2020
	Paralichthys isosceles	Brazil	KU314822	Fonseca et al., 2019
	Stenella clymene	Argentina	MW724483	Hernández-Orts et al., 2021
	Arctocephalus australis	Argentina	MF497333	Hernández-Orts et al., 2017a
	Spheniscus magellanicus	Brazil	MF497335	Hernández-Orts et al., 2017a
	Otaria flavescens	Argentina	KX957714, MF497334	Hernández-Orts et al., 2017a, b
	Paralichthys adspersus	Peru	MZ920052–MZ920055	Mondragon-Martinez et al., 2021^*
	Paralabrax humeralis	Peru	MZ920056–MZ920059	Mondragon-Martinez et al., 2021^*
	Cheilodactylus variegatus	Peru	MZ920060–MZ920063	Mondragon-Martinez et al., 2021^*
	Otaria byronia	Peru	MZ920064–MZ920067	Mondragon-Martinez et al., 2021^*
Corynosoma hannae	Colistium guntheri	New Zealand	KX957724, KX957725	Hernández-Orts et al., 2017b
	Leucocarbo chalconotus	New Zealand	KX957718–KX957721, KX957723	Hernández-Orts et al., 2017b
	Phalacrocorax punctatus	New Zealand	KX957722	Hernández-Orts et al., 2017b
	Phocarctos hookeri	New Zealand	KX957715–KX957717, JX442191	Hernández-Orts et al., 2017b; García-Varela et al., 2013
	Peltorhamphus novaezeelandiae	New Zealand	KY909260–KY909263	Anglade & Randhawa, 2018
Corynosoma semerme	Halichoerus grypus	Germany	MF001277	Waindok et al., 2018
Corynosoma semerme	Callorhinus ursinus	USA	MK119253	Lisitsyna et al., 2019
Corynosoma obtuscens	Halichoerus grypus	New Zealand	JX442192	García-Varela et al., 2013
Corynosoma villosum	Callorhinus ursinus	USA	MK119251	Lisitsyna et al., 2019
Corynosoma validum	Callorhinus ursinus	USA	MK119252	Lisitsyna et al., 2019
Corynosoma enhydri	Enhydra lutris	USA	DQ089719	García-Varela & Nadler, 2006
Corynosoma magdaleni	Phoca vitulina	Germany	MF078642	Waindok et al., 2018
Corynosoma nortmeri	Phoca vitulina	Germany	MF001278	Waindok et al., 2018
Corynosoma strumosum	Phoca vitulina	USA	EF467870	García-Varela & Pérez-Ponce de León, 2008
	Pusa hispida botnica	Finland	EF467871	García-Varela & Pérez-Ponce de León, 2008
	Zalophus californianus	USA	MK119250	Lisitsyna et al., 2019
Bolbosoma balaenae	Balaenoptera physalus	Italy	MZ047281	Santoro et al., 2021
Bolbosoma turbinella	Eschrichtius robustus	USA	JX442189	García-Varela et al., 2013
Andracantha phalacrocoracis	Zalophus californianus	USA	MK119254	Lisitsyna et al., 2019
Hexaglandula corynosoma	Nyctanassa violacea	Mexico	EU189488	Guillén-Hernández et al., 2008
Pseudocorynosoma anatarium	Bucephala albeola	Mexico	KX688148	García-Varela et al., 2017
Pseudocorynosoma tepehuanesi	Oxyura jamaicensis	Mexico	KX688139	García-Varela et al., 2017
Polymorphus obtusus	Aythya affinis	Mexico	JX442195	García-Varela et al., 2013
Profilicollis bullocki	Emerita analoga	Mexico	JX442197	García-Varela et al., 2013
Profilicollis chasmagnathi	Oligosarcus jenynsii	Argentina	MT580124	Levy et al., 2020
Polymorphus trochus	Fulica americana	Mexico	JX442196	García-Varela et al., 2013

Data for the population of Corynosoma australe used in the haplotype networking using mt Cox1 gene_ Asterisk shows sequences unpublished on NCBI database_ Newly generated sequences are presented in bold_

Geographical Locality^*	Cox1Haplotype no.	ID in Fig. 46	GenBank accession nos.	References
Pacific coast near San Francisco, California, USA	H1	U1	ON619618, ON614719	Present study
Sausalito, California, USA	H2	U	MK119245-MK119249	Lisitsyna et al. 2019
Baja California, Mexico	H2	M	MT676816-MT676818	García-Varela et al. 2021
Rio de Janeiro, Brazil	H3	B	KU314822	Fonseca et al. 2019
Chubut, Argentina	H4	A	MW724483	Hernández-Orts et al. 2021
Baja California, Mexico	H5	M	MT676813	García-Varela et al. 2021
Sonora, Mexico	H6	M	MT676811	García-Varela et al. 2021
Baja California, Mexico	H7	M	MT676814	García-Varela et al. 2021
Sonora, Mexico	H8	M	MT676810	García-Varela et al. 2021
Northern Patagonia, Argentina	H9	A	MT676821	García-Varela et al. 2021
Sonora, Mexico	H10	M	MT676809	García-Varela et al. 2021
Baja California Sur, Mexico	H11	M	MT676808	García-Varela et al. 2021
Northern Patagonia, Argentina	H12	A	MT676823	García-Varela et al. 2021
Northern Patagonia, Argentina	H13	A	MT676819, MT676820, MT676822	García-Varela et al. 2021
Baja California, Mexico	H14	M	MT676815	García-Varela et al. 2021
Northern Patagonia, Argentina	H15	A	MT676824	García-Varela et al. 2021
Northern Patagonia, Argentina	H16	A	MF497333	Hernández-Orts et al. 2021
Baja California, Mexico	H17	M	MT676812	García-Varela et al. 2021
Rio de Janeiro, Brazil	H18	B	MF497335	Hernández-Orts et al. 2021
Northern Patagonia, Argentina	H19	A	MF497334	Hernández-Orts et al. 2021
Northern Patagonia, Argentina	H20	A	KX957714	Fonseca et al. 2019
Peru	H21	P	MZ920052, MZ920053, MZ920055, MZ920056-MZ920062, MZ920064-MZ920067	Mondragon-Martinez et al. 2021^*
Peru	H22	P	MZ920054, MZ920063	Mondragon-Martinez et al. 2021^*

Chemical composition of trunk spines and eggs of Corynosoma australe from Zalophus californianus in California_

Elements^*	Spines (longitudinal sections)			Eggs (cross sections)

	Anterior	Middle	Posterior	Edge (shell)	Center (acanthor)
Magnesium (Mg)	0.00	0.00	0.09	0.00	0.50
Sodium (Na)	0.00	0.00	0.00	0.00	0.00
Phosphorous (P)	1.39	0.00	1.16	0.91	7.73
Sulfur (S)	12.64	5.97	15.61	0	2.30
Calcium (Ca)	1.64	1.07	1.96	1.34	3.41

List of acanthocephalan species used for phylogenetic analysis based on the 18S rDNA gene sequences_ Newly generated sequences are presented in bold, NA=host name not available_

Species	Host	Host origin	GenBank accession nos.	References
Corynosoma australe	Zalophus californianus	USA	ON614192, ON614199	present study
	Zalophus californianus	USA	MK119255	Lisitsyna et al., 2019
	Phocarctos hookeri	Mexico	JX442168	García-Varela et al., 2013
Corynosoma obtuscens	Callorhinus ursinus	Mexico	JX442169	García-Varela et al., 2013
Corynosoma validum	Callorhinus ursinus	Mexico	JX442170	García-Varela et al., 2013
Corynosoma enhydri	NA	USA	AF001837	Near et al., 1998
Corynosoma magdaleni	Phoca hispida botnica	Mexico	EU267803	García-Varela et al., 2009
Corynosoma strumosum	Phoca vitulina	Mexico	EU267804	García-Varela et al., 2009
Bolbosoma balaenae	Nyctiphanes couchii	Spain	JQ040306	Gregory et al., 2011^*
Bolbosoma turbinella	Eschrichtius robustus	Mexico	JX442166	García-Varela et al., 2013
Bolbosoma vasculosum	Lepturacanthus savala	Indonesia	JX014225	Verweyen et al., 2011
Andracantha gravida	Phalacrocorax auritus	Mexico	EU267802	García-Varela et al., 2009
Hexaglandula corynosoma	Nyctanassa violacea	Mexico	EU267808	García-Varela et al., 2009
Pseudocorynosoma anatarium	Bucephala albeola	Mexico	EU267801	García-Varela et al., 2009
Pseudocorynosoma constrictum	Anas clypeata	Mexico	EU267800	García-Varela et al., 2009
Profilicollis bullocki	Emerita analoga	Mexico	JX442174	García-Varela et al., 2013
Profilicollis botulus	Somateria mollissima	Mexico	EU267805	García-Varela et al., 2009
Polymorphus trochus	Fulica americana	Mexico	JX442196	García-Varela et al., 2013

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

Figures & Tables

Figs. 1, 2.

Figs. 3–8.

Figs. 9–14.

Figs. 15–20.

Figs. 21–26.

Figs. 27–32.

Figs. 33–36.

Figs. 37–39.

Fig. 40.

Fig. 41.

Fig. 42.

Fig. 43.

Chemical composition and localization of elements in hooks of Corynosoma australe from Zalophus californianus in California_

Chronological taxonomic history of Corynosoma australe from marine mammals, with special reference to ventral trunk spines in females_

List of acanthocephalan species used for phylogenetic analysis based on the mt Cox1 gene sequences_ Newly generated sequences are presented in bold_

Data for the population of Corynosoma australe used in the haplotype networking using mt Cox1 gene_ Asterisk shows sequences unpublished on NCBI database_ Newly generated sequences are presented in bold_

Chemical composition of trunk spines and eggs of Corynosoma australe from Zalophus californianus in California_

List of acanthocephalan species used for phylogenetic analysis based on the 18S rDNA gene sequences_ Newly generated sequences are presented in bold, NA=host name not available_

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