“Reviving museum oddities: rehydrating Trematodes curiosities!” A successful method for rehydrating dried museum specimens for a revision of the genus Octangium (Trematoda), parasites of green sea turtle Chelonia mydas

doi:10.21203/rs.3.rs-5018739/v1

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“Reviving museum oddities: rehydrating Trematodes curiosities!” A successful method for rehydrating dried museum specimens for a revision of the genus Octangium (Trematoda), parasites of green sea turtle Chelonia mydas

https://doi.org/10.21203/rs.3.rs-5018739/v1

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Octangium Looss, 1902 is a taxonomically challenging genus of microscaphidiid trematodes, with members harmful at the adult stage to their reptile hosts, the turtles. In Arthur Loss’s collection preserved at the Swedish Museum of Natural History, we found mounted slides of O. sagitta (Looss, 1899) and of O. hasta Looss, 1902, and vials containing Octangium spp. preserved in ethanol from a host and locality matching those listed in Looss (1899), suggesting that this is the part of type material. Unfortunately, several specimens were dried. We attempted herein to rehydrate them, make whole stained mounts, and investigate the material.

Additionally, we examined several specimens of O. sagitta and O. hasta from the Invertebrate collections at the SMNH and provided illustrated redescriptions. In light of the available data, also discuss records of O. sagitta, O. hasta, and previous species synonymized with O. sagitta and provide a key for the identification of Octangium spp. We herein reinstate Octangium takanoi Kobayashi, 1921 and O. hasta, and amend Octangium based on a re-examination of adult specimens of the type material of O. sagitta [type species] and of O. hasta, infecting the intestine of the green sea turtle Chelonia mydas. We demonstrate that O. takanoi differs from O. sagitta by being twice smaller, by the shape of the oral sucker (rectangular vs. rounded), and especially by the extent of vitellaria, with their anterior end situated at the level of the anterior margin of the testes in O. takanoi, while they extend to the intestinal bifurcation in O. sagitta. Additionally, they can be easily distinguished by more anterior placing of the testes in O. takanoi. Similarly, O. hasta can be easily distinguished from O. sagitta by being significantly smaller in all body measurements. More importantly, O. sagitta and O. hasta can be easily distinguished by the shape of the oral sucker. We refute thus the synonymies between O. sagitta and the two species O. takanoi and O. hasta, and we reinstate the two latter species as valid species. We summarized and mapped the distribution of current valid species of the genus Octangium.

Parasitology

Octangium

Chelonia mydas

Arthur Looss

Collection

Naturhistoriska riksmuseet

Egypt

Natural History museums worldwide, in addition to housing billions of relevant specimens offering an exceptional potential parasitological dataset for taxonomical studies and various other purposes (Wood & Vanhove, 2023), play a crucial role as a “biodiversity” resource, not only for taxonomists but also for scientists who are dependent on accurate information that transcends specific disciplines. Additionally, as global ecosystems undergo rapid transformation, there is a significant risk of losing biodiversity information. This includes insights into the impacts of accelerated ecological change, which in turn affect human health, the sustainability of food resources, conservation efforts, and the integrity of natural systems (Hoberg et al., 2009). Moreover, collections can be considered historical and environmental benchmarks, allowing us to evaluate shifts in faunal structure. An applied aspect of this is the monitoring of invasive species and emerging diseases resulting from processes such as global climate warming, as well as other human-induced and natural phenomena.

In the field of parasitology, it is not uncommon to encounter perplexity regarding taxonomy, the host, and geographic ranges of species. To address these challenges, natural history collections play a key role as they include invaluable type series and vouchers documenting associations across different temporal and spatial scales. They form thus the cornerstone of systematic research by establishing an empirical record that enhances our comprehension of the parasites as part of the biosphere. Moreover, collections are increasingly vital for the advancement of biodiversity informatics, consolidating knowledge on the identity, geographic distribution, ecology, phylogeny, population structure, and historical context of these pathogens’ diversity (Hoberg et al., 2009).

Systematics and specimens-based collections are the foundation of an important part of what has been accomplished in parasitology and have resolved a substantial number of real-world problems in human and animal health (Brooks & Hoberg, 2000). Collections can serve as invaluable resources for public health responders seeking to identify the origins and distribution of disease agents. For example, remarkable success has been achieved in protecting human populations from Guinea Worm (Nematoda) once all its developmental stages and host species were sampled, preserved, documented, and analyzed. Moreover, understanding the pathways of tropical diseases like elephantiasis (caused by a Nematode), and schistosomiasis (caused by a Trematode) through various hosts requires the creation and examination of populations of potential host and vector species, both wild and domesticated. Hence, access to collections and the insights gleaned from their analyses will be crucial in comprehending and disrupting the cycles of disease transmission involving multiple species (DiEuliis et al., 2016).

The Swedish Museum of Natural History (SMNH) houses a large collection of parasitic invertebrates, including part of the collections of the eminent parasitologist Professor Arthur Looss (Kuzmina & Holovachov, 2023). This collection includes several slides that are type material, and vials containing trematodes preserved in ethanol, along with several publication-ready and original line drawings (Bouguerche et al., 2023) along with notes on host and locality (Fig. 1. A-C).

During an ongoing investigation on the systematics of Trematodes at the SMNH collections, we found vials containing Octangium spp. preserved in ethanol from a host and locality matching those listed in Looss (1899) suggesting that this is the part of type material used by Looss in his study. Unfortunately, several specimens were dried out (Fig. 1. D, E). Some of the line drawings of parasitic Trematodes that we had, matched the plates given in Looss’s work on systematics and nomenclature of trematodes from sea turtles (Looss, 1902) (Fig. 2) indicating that this is part of the type material.

Given their host, this collection of parasites of turtles certainly warrants more attention. Sea turtles confront a multitude of threats, including pollution, habitat degradation, the impacts of climate change, interactions with fisheries, and ingestion of marine debris (Duncan et al., 2019). Moreover, they experience intussusception, a condition where a section of the gastrointestinal tract (known as the intussusceptum) invaginates into the lumen of an adjacent portion, termed the intussuscipiens (MacPhail, 2002) resulting in partial or total obstruction of the gastrointestinal tract (Levit & Bauer, 1992). Interestingly, the existence of parasites is frequently regarded as a predisposing factor for intussusception (see (Duncan et al., 2019). Moreover, Duncan et al. (2019) demonstrated that parasitic infestation by Octangium spp. in sea turtles can root intussusception, subsequently leading to the death of the host turtle.

Timely diagnosis is crucial for achieving a favorable prognosis and successful rehabilitation of the host and can only be preceded by an acute identification of the causal Trematode. Thus, we attempted to rehydrate the Trematodes Octangium sp. found in the SMNH, make whole stained mounts, and investigate the material. Additionally, we examined several specimens of O. sagitta and O. hasta from the Invertebrate collections at the SMNH and provided detailed illustrated redescriptions. We also discuss records of O. sagitta, O. hasta, and previous species synonymized with O. sagitta and provide a key for the identification of Octangium spp.

2.1. Rehydration of dried specimens

The current study is based solely on A. Looss’s specimens and archival documents preserved in the archives and general-type collections of the Department of Zoology, Swedish Museum of Natural History. Several Trematodes, with notes on the host, locality, and microhabitat in the host were dried out. For the rehydration of dried brittle microscaphidiids from A. Looss’s collection, we followed the protocol of Van Cleave & Ross (1947) using trisodium phosphate to soften specimens prior to staining. Hard, brittle, and unyielding dry Trematodes were passed down the grades of alcohol to water and were then placed in a solution of approximately 0,25 percent of trisodium phosphate in distilled water until softened. Specimens were kept in the trisodium phosphate solution (T-Ph-S) for several hours or even days, from four hours to two weeks. No warming oven was used to attain the proper degree of softness and pliability.

Treated specimens in T-Ph-S were kept under observation until obtaining the desired degree of softness and translucency. One specimen was treated separately in a petri dish under a dissecting microscope, to check the progress of the treatment. Treated specimens are kept in the solution until turning from excessive hard dark brownish specimens to translucid soft specimens.

When the desired degree of softness and translucency has been reached, the treated specimens are removed from the T-Ph-S to distilled water to check the action. When softened, the treated thus treated are washed several times in distilled water, then stored in 70 ethanol.

2.2. Staining and slides mounting

Whole-mounts for morphological analysis were stained with acetocarmine for 10 minutes, dehydrated in a graded ethanol series (70, 96. 100. For 20 minutes each), cleared carefully in clove oil, and mounted in Canada balsam. Drawings were made through a Nikon Eclipse i80 microscope with DIC (differential interference contrast) and a drawing tube. Drawings were scanned and redrawn on a computer with Adobe Illustrator 2023.

Measurements of whole-mounts are in micrometres and indicated as the range followed by the

number of measurements in parentheses. The maximum width was taken slightly behind the middle of the body following Looss (1899). The following abbreviation is used: SMNH, Swedish Museum of Natural History, Stockholm, Sweden.

2.3. Examination of comparative material

The collection also includes several total mounts and sections, prepared as described in respective original descriptions of Octangium spp (Looss, 1899, 1901, 1902). Additional material examined in this study includes stained mounts of O. sagitta and O. hasta from Looss’s collection found in the Trematodes collection at the SMNH that were examined and studied for comparison. The studied comparative material included four whole-mounts of O. sagitta (15 specimens) ex C. mydas from Cairo and from Alexandria, Egypt, Eastern Mediterranean; 7 sections of O. sagitta ex C. mydas from Cairo, Egypt, Eastern Mediterranean; 7 whole-mounts of O. hasta (51 specimens) from ex C. mydas from Cairo, Egypt, Eastern Mediterranean; 3 sections ex C. mydas from Cairo, Egypt, Eastern Mediterranean. Measurements and drawings are tacking as explained above. Measurements of specimens from the SMNH Trematodes’ collection were provided separately.

Family Microscaphidiidae Looss, 1900

Genus Octangium Looss, 1902

Octangium sagitta (Looss, 1899) Looss, 1902

Accepted name

Octangium sagitta (Looss, 1899) Looss, 1902

Unaccepted name/ Synonyms

Microscapha sagitta Looss, 1899 (Superseded combination). Microscaphidium sagitta (Looss, 189) Looss, 1901 (Superseded combination). Octangium hasta Looss, 1902 (Tentative synonymy by Blair (1987) refuted). Octangium takanoi Kobayashi, 1921 (Tentative synonymy by Blair (1987) refuted).

Original name

Microscapha sagitta Looss, 1899.

Type-host: Chelonia mydas (Testudines: Cheloniidae), the green sea turtle (Looss, 1899).

Type-locality

off Egypt, Eastern Mediterranean (Looss, 1899).

Site in host in original description

Rectum (Looss, 1899).

Additional hosts: Caretta caretta (Cheloniidae), the Loggerhead sea turtle (Marangi et al., 2020). Eretmochelys imbricata (Cheloniidae), the Hawksbill turtle (Dyer et al., 1995)

Additional localities

Italy, Adriatic Sea and Northern Ionian Sea, Central Mediterranean (Marangi et al., 2020). Florida, USA, Atlantic, Western-Central (Wyneken et al., 2013). Puerto Rico, Atlantic, Western-Central (Dyer et al., 1991; Dyer et al., 1995). Rio De Janeiro, Brazil, Atlantic, Southwest (Cavaco, 2021; Silva, 2020). Gulf of Mexico, USA, Atlantic, Western-Central (Overstreet et al., 2009). Queensland, Australia, Pacific, Western Central (Johnston, 1913; Johnston, 1916)

Other reported habitats

the large intestine (Cavaco, 2021; Dyer et al., 1991; Looss, 1902; Overstreet et al., 2009). Terminal section of the small intestine) (Looss, 1902; Overstreet et al., 2009). Gastrointestinal tract (Silva, 2020). Intestine (Johnston, 1913). Note that the report of Marangi et al. (2020) was based on faecal examination to detect eggs in a faecal sample and thus the location of adults in the corresponding host is not available. Habitat in the record by Wyneken et al. (2013) and that of Dyer et al. (1995) not available.

Specimens deposited: specimens examined for morphological study, whole mounts: rehydrated specimens from A. Looss’s dried specimens’ collection at the SMNH: O. sagitta ex Chelonia mydas from Cairo, Egypt, Eastern Mediterranean, rehydrated, stained and mounted (SMNH type 9690–9715)

Material examined for comparison:

Whole mounts, from A. Looss’s collection at the SMNH: O. sagitta ex Chelonia mydas from Alexandria, Egypt, Eastern Mediterranean (SMNH 140048) marked as juveniles (see discussion); O. sagitta ex C. mydas from Alexandria, Egypt, Eastern Mediterranean (SMNH 140049); O. sagitta ex C. mydas from Cairo (Abuk), Egypt, Eastern Mediterranean (SMNH 140050, 140051), O. sagitta ex C. mydas from Cairo, Egypt, Eastern Mediterranean (SMNH 140052).

Sectioned material, from A. Looss’s collection at the SMNH

ex O. sagitta from Chelonia mydas from Egypt, Eastern Mediterranean; 2 undefined sections (SMNH 140053, SMNH 140059), 2 “queer” sections (SMNH 140054140055), 3 sagittal sections (SMNH 140056, SMNH 140058),

3.1. Redescription (Figs. 3, 4)

Comparative measurements in Tables 1, 2. Body stocky, ending posteriorly with 2 visible caudolateral projections separated by a terminal notch. Tegument thick, smooth. Mouth terminal.

Table 1

Hosts and localities of *Octangium* spp. Rej., rejected synonym. ¹ occasionally small intestine. 2 currently *taxon inquirendum*.
Species	Synonyms	Type-host	Microhabitat	Type locality
Octangium sagitta (Looss, 1899)	Microscapha sagitta Looss, 1899 Microscaphidium sagitta (Looss, 1899) Octangium hasta Looss, 1902 Rej. Octangium takanoi Kobayashi, 1921 Rej.	Chelone mydas	Rectum	Egypt, EM	(Blair, 1987; Looss, 1899, 1901, 1902)
Octangium hasta Looss, 1902	Octangium sagitta Rej.	Chelone mydas	Large intestine ¹	Egypt, EM	(Blair, 1987; Looss, 1902)
Octangium takanoi Kobayashi, 1921	Octangium sagitta Rej.	Chelone mydas	Intestine	Singapore, WCP	(Kobayashi, 1921)
Octangium elongatum Chattopadhyaya, 1972		Chelone mydas	Large intestine	India, EIO	(Chattopadhyaya, 1972)
Octangium microrchis Chattopadhyaya, 1972		Eretmochelys imbricata	Large intestine	India, EIO	(Chattopadhyaya, 1972)
Octangium hyphalum Blair, 1987		Chelone mydas	Pseudocaecum	Australia, WCP	(Blair, 1987)
Abbreviations: EIO., Eastern Indian Ocean. EM., Eastern Mediterranean. WCP., Western Central Pacific

Table 2

Comparative measurements for *Octangium sagitta* (Looss, 1899) from the Mediterranean. * Diameter. ^a Looss (1899) also reported a length of 1000–2000 µm for very young specimens. ^b exceptionally 8. NA, not available.
Habitat	Rectum	Intestine	ED	ED	ED	NA	NA
Locality	Egypt, E. M.	-	Alexandria, Egypt, E. M.	Cairo, Egypt, E. M.	Egypt, E. M.	Egypt, E. M.	Egypt, E. M.
Number of specimens	2 adults	-	1 adult	13 adults	23 adults	9	14
Reference	Looss (1899)	Looss (1902)	Present study	Present study	Present study, rehydrated specimens	Sey (1977) in (Blair, 1987)	Looss’s (1902) type material from NHR collections (Blair, 1987)
Body length	7 000 ^a	Up to 10 000	6776	7061 (4231–10250, n = 11)	6711 (1575–7863, n = 21)	6560 (4900–7800)	6970 (4300–10 380)
Body width	2 000	Largest 2 000	1922	1916 (1472–2424, n = 11)	1215 (834–1537, n = 21)	2100 (1660–2420)	1980 (1500–2500)
Oral sucker length			276	313 (248–360, n = 9)	321 (237–377, n = 23)	350 (290–480)	310 (230–360, n = 13)
Oral sucker width	330 *	370	358	432 (337–518, n = 9)	300 (220–361, n = 23)	340 (280–400)	410 (340–520, n = 13)
Oesophagus length			1434		1844 (1197–2180, n = 17)	1440 (1210–1660)	1590 (900–2400)
Distance anterior end- intestinal bifurcation			1815	1867 (1249–2411, n = 11)	2179 (1422–2576, n = 18)
Distance anterior testes- intestinal bifurcation			1145	1112 (417–1773, n = 10)	859 (655–1052, n = 9)	780 (480–1000)	810 (400–1400)
Anterior testis length				860 (45 − 1174, n = 12)	648 (426–837, n = 7)	760 (500–870)	870 (450–1140, n = 13)
Anterior testis width				663 (186–1068, n = 12)	307 (239–375, n = 7)	690 (410–1020)	660 (210–1150, n = 13)
Posterior testis length			1018	885 (471–1243, n = 12)	641 (421–780, n = 7)	750 (590–950)	870 (500–1210, n = 12)
Posterior testis width			630	649 (194–1043, n = 12)	307 (227–398, n = 7)	670 (560–870)	740 (230–1220, n = 12)
Ovary length			272	373 (250–521, n = 5)	137 (122–170, n = 4)	270 (160–320, n = 7)	280 (130–440, n = 11)
Ovary width			203	293 (230–454, n = 5)	154 (115–193, n = 4)	240 (130–310, n = 7)	240 (100–360, n = 12)
Number of excretory diverticula	6–7 ^b
Eggs length	80	80	80	77 (70–81, n = 11)	78 (72–86, n = 23) 49 (45–55, n = 23)	79–87	75–85
Eggs width	53	50	54	51 (42–56, n = 11)		56–64	48–60
Abbreviations: NA, not available. E.M., Eastern Mediterranean. SW.A. Southwest Atlantic.

Oral suckers small, rounded to elliptical, with a pair of intramural diverticula distally. Oesophagus long, straight, thick-walled, with an anterior thickening of its musculature at posterior level of oral sucker. Oesophagus ending posteriorly with a voluminous muscular bulb, straight or almost so, with distinct muscular bulb at posterior end. Esophageal bulb with a wide visible lumen, surrounded by numerous glandular cells. Intestinal bifurcation anterior to tests. Caeca large, thick-walled, visibly wrinkled in most specimens, ending close posterior end of body.

Testes intercaecal in middle third of body, tandem. Anterior testis always posterior to intestinal bifurcation. Seminal vesicle immediately posterior to, elongated, slightly sinuous in some parts, ending anteriorly by a thin ejaculatory duct. Hermaphroditic duct not examined. Genital pore posterior to sucker.

Ovary small, always posterior to testes. Oviduct thin, extended by a large uterus. Uterus extending anteriorly and ending by a thin metraterm. Metraterm joining ejaculatory duct close to genital pore. Vitellarium follicular, arranged in two lateral fields and one median field. Lateral vitelline fields not co-existing entirely with caeca, extending from level of intestinal bifurcation, posteriorly before ends of caeca. Median vitelline field extending from posterior level of ovary to posterior ends of caeca. Eggs in operculated.

Excretory rosette formed of 5–8 diverticula.

Octangium hasta Looss, 1902

Accepted name

Octangium hasta Looss, 1902

Unaccepted name/ Synonyms

Octangium sagitta (Looss, 1899) Looss, 1902 (Tentative synonymy by Blair (1987) refuted)

Original name

Octangium hasta Looss, 1902.

Type-host: Chelonia mydas (Testudines: Cheloniidae), the green sea turtle (Looss, 1902).

Type-locality

Cairo, Egypt, Eastern Mediterranean (Looss, 1902).

Site in host in original description

Large intestine (Looss, 1902).

Specimens examined for morphological study:

Whole mounts, from A. Looss’s collection at the SMNH: O. hasta ex C. mydas from Alexandria, Egypt, Eastern Mediterranean (SMNH 140048) marked as juveniles (see discussion); O. hasta ex C. mydas from Cairo, Egypt, Eastern Mediterranean (SMNH 140038–140044).

Sections, from A. Looss’s collection at the SMNH: O. hasta ex C. mydas from Cairo, Egypt, Eastern Mediterranean (SMNH 140045–140047).

1.1. Redescription (Figs. 5, 6)

Measurements in Table 3. Body elongate, maximum width at level of midbody. Width uniform in main part of the body, narrowing anteriorly and posteriorly. Body ending posteriorly with 2 caudolateral projections) separated by a terminal notch. Tegument thick, smooth. Mouth terminal.

Table 3

Comparative measurements for *Octangium sagitta* (Looss, 1899) from the Pacific. * Diameter. ^A Looss (1899) also reported a length of 1000–2000 µm for very young specimens. ^bexceptionally 8.
Habitat	ED	ED	ED	NA	NA	Large intestine	Gastrointestinal tract
Locality	Alexandria, Egypt, EM.	Cairo, Egypt, EM.	Egypt, EM.	Queensland, WCP	Puerto Rico, WCA	Brazil, SWA.	Brazil, SWA.
Number of specimens	1 adult	13 adults	23 adults	11	15	NA	NA
Reference	Present study	Present study	Present study, rehydrated specimens	(Blair, 1987)	Fischthal & Acholonu 1976 (Blair, 1987)	Cavaco (2021)	(Silva, 2020)
Body length	6776	7061 (4231–10250, n = 11)	6711 (1575–7863, n = 21)	8440 (6140–10 800)	5130 (4000–6000)	6000 (4540–7690)	5580–6470
Body width	1922	1916 (1472–2424, n = 11)	1215 (834–1537, n = 21)	2480 (1880–2780)	1350 (1100 − 1540)	1700 (990–2980)	3760–4310
Oral sucker length	276	313 (248–360, n = 9)	321 (237–377, n = 23)	510 (300–630, n = 9)	320 (260–340)	237 (158–351)	510–580
Oral sucker width	358	432 (337–518, n = 9)	300 (220–361, n = 23)	520 (450–590, n = 9)	270 (220–320)	285 (214–377)	530–630
Oesophagus length	1434		1844 (1197–2180, n = 17)	1780 (1320–2400)	1380 (1160– 1520)	923 (490–1269)	1020–1270
Distance anterior end- intestinal bifurcation	1815	1867 (1249–2411, n = 11)	2179 (1422–2576, n = 18)				280–310
Distance anterior testes- intestinal bifurcation	1145	1112 (417–1773, n = 10)	859 (655–1052, n = 9)	960 (740–1400)	500 (320–580)
Anterior testis length		860 (45 − 1174, n = 12)	648 (426–837, n = 7)	1030 (650–1430)	450 (280–580)	667 (381–902)	500–610
Anterior testis width		663 (186–1068, n = 12)	307 (239–375, n = 7)	750 (560–1150)	230 (160–340)	666 (383–994)	950–1090
Posterior testis length	1018	885 (471–1243, n = 12)	641 (421–780, n = 7)	1080 (680–1380)	440 (290–530)	628 (468–912)	520–580
Posterior testis width	630	649 (194–1043, n = 12)	307 (227–398, n = 7)	840 (590–1360)	230 (140–350)	489 (284–755)	960–1110
Ovary length	272	373 (250–521, n = 5)	137 (122–170, n = 4)	310 (210–370)	130 (90–180)	206 (146–258)	450–510
Ovary width	203	293 (230–454, n = 5)	154 (115–193, n = 4)	290 (150–380)	70 (60–110)	176 (99–245)	290–320
Number of excretory diverticula
Eggs length	80	77 (70–81, n = 11)	78 (72–86, n = 23) 49 (45–55, n = 23)	(71–83, n = 12)	67–75	83 (71–92)
Eggs width	54	51 (42–56, n = 11)		(48–52, n = 12)	39–50	49 (42–57)
Abbreviations: NA, not available. E.M., Eastern Mediterranean. WCA., Western-central Atlantic. WCP., Western central pacific. SWA. Southwest Atlantic.

Oral sucker somewhat rectangular. Oesophagus long, thick walled, with a wide lumen, ending posteriorly with a distinct oesophageal bulb. Esophageal bulb with a wide lumen, surrounded by numerous glandular cells. Intestinal bifurcation at anterior third. Caeca straight.

Testes pre-ovarian, intercaecal in midbody, posterior to intestinal bifurcation, oval, disposed in tandem. Eggs in utero operculated. Seminal vesicle posterior to anterior tests. Ejaculatory duct joining metraterm. Hermaphroditic duct not examined. Genital pore posterior to ventral sucker.

Ovary small, ventral, always posterior to testes. Oviduct thin, extended by the uterus. Uterus extending anteriorly and ending by a thin metraterm. Vitellarium arranged as in O. sagitta. Lateral vitelline co-existing partially with caeca, anterior limit of lateral vitelline fields not attending level of intestinal bifurcation, posterior limit before ends of caeca. Median vitelline field extending from posterior level of ovary and not reaching posterior ends of caeca. Vitellarium follicular, arranged in two lateral fields and one median field. Lateral vitelline fields not co-existing entirely with caeca, extending from level of intestinal bifurcation, posteriorly before ends of caeca. Median vitelline field extending from posterior level of ovary to posterior ends of caeca. Eggs in operculated.

2.1. Looss’s Trematodes collection

Arthur Looss died after a long illness on May 4, 1923, at Giessen. Born on May 16, 1861, at Chemnitz, he received his education in his native city and at Lodz in Poland, then at the University of Leipzig where he received the degree of Doctor of Philosophy, his thesis being on the Trematodes (Vaughan, 1924). He was for many years, a professor of parasitology and biology at the School of Medicine in Cairo. He pursued his work until after the outburst of the World War when he retired from his chair and returned to Germany (Vaughan, 1924).

Looss dedicated his life to the study of helminthology, not only systematics and nomenclature as he made outstanding inputs to the medical aspect of helminthology. One of the spectacular discoveries of his career, was when he was sent by Leuckart to Egypt to study the transmission of bilharzia, where there he became interested in hookworm and due to an accidental infection of himself that made the discovery of the aptitude of larval worm to enter the skin, then transported to the heart, lungs and intestinal tract (Vaughan, 1924). Certainly, by the finding of the mode of infection Looss also clearly helped determine the way to prevent the disease. Hence, it is only fair to say that Looss not only “made for himself a name as one of the most distinguished of helminthologists” but also “put the world under a debt of gratitude to him”.

After his death, his collection was sold to different museum and part of it to the Swedish Museum of Natural history (Kuzmina & Holovachov, 2023). This collection includes numerous slides that are actually type material, and vials comprising ethanol-preserved trematodes, and various publication-ready and original line drawings (Bouguerche et al., 2023) beside notes of records on host and locality. For O. sagitta, some of the line drawings of parasitic Trematodes that we had, matched the plates given in Looss’s work on systematics and nomenclature of trematodes from sea turtles (Looss, 1902) (Fig. 2) indicating that this is part of the type material.

Thus, we attempted to rehydrate the Trematodes O. sagitta and we made whole stained mounts that we used to investigate these Trematodes and the systematics of the genus. Hence, these Trematodes even in compromised condition, provided still instrumental data for the systematics of the genus Octangium, aiding in the identification of Octangium spp. Advances in genetic and molecular techniques will certainly also allow to extraction of DNA from degraded specimens, offering insights into evolutionary relationships and the genetic structure of populations.

2.2. Rehydration of dried specimens

Van Cleave and Ross (1947) stated that freshly preserved specimens become soft and translucent shortly after, while hard dry specimens soften after several hours or even days. Herein, all the microscaphidiids from A. Looss’s collection were hard, brittle, and unyielding after long preservation and thus required a longer treatment up to four hours in the rehydrating solution. However, smaller Trematodes, such as Lecithocladium excisum (Rudolphi, 1819) (in an upcoming study) required shorter rehydrating time and became softened within 45 minutes at the earliest. Van Cleave and Ross (1947) also mentioned that hard dry specimens might require even days in a warming oven before they attain the proper degree of softness and pliability. However, no warming oven was used to attain the proper degree of softness and pliability. The only difference was examined regarding the time of rehydration. Specimens that were kept for several days in the rehydrating solution tend to clear better in clove oil after the staining.

2.3. Systematics of Octangium spp.

Microscaphidiid Trematodes of the genus Octangium Looss, 1902 are typically parasites of the intestine of marine turtles (Cheloniidae), in nearly all tropical and subtropical seas. Members of this genus are readily distinguished by a body with a profound posterior terminal notch, visible between paired caudolateral projections, a simple not recurved excretory vesicle, and the presence of a rosette of diverticula present just proximal to the excretory pore (Blair, 2005).

The genus Octangium is in need of systematic revision. Overall, 7 species have been assigned to the genus Octangium (WoRMS, 2023): O. sagitta of which O. hasta, and O. takanoi Kobayashi, 1921, were considered synonyms; and O. hyphalum Blair, 1987, of which O. takanoi Kobayashi sensu Mehrotra (1973) and Tandon and Gupta (1981) was considered a synonym (Blair, 2005). The most recent species are O. elongatum Chattopadhyaya, 1972, from the large intestine of the green sea turtle Chelonia mydas and O. microrchis Chattopadhyaya, 1972 from the hawksbill sea turtle Eretmochelys imbricata from the eastern Indian Ocean, off India, are both currently taxon inquirendum (Blair, 1987). Another Microscaphidiid species from marine turtles assigned previously to this genus is O. travassosi (Ruiz, 1943), first described from the intestine of an unidentified marine turtle referred to as “Tartaruga marinha (sp.?)” from the southwest Atlantic, off Brazil as Neoctangium travassosi Ruiz, 1943 (Ruiz, 1943), then placed in the genus Octangium when Yamaguti (1958) synonymized Neoctangium with Octangium, first without explanation (Blair, 1987), then justified his decision and wrote “Ruiz (1943) distinguished Neoctangium Ruiz, 1943 from Octangium simply because of the presence of 4 pairs of cephalic [sic] papillae” (Yamaguti, 1971). Blair (1987) refuted the synonymy between the two genera and considered it “unreasonable” as Ruiz (1943) omitted any mention of the excretory system in his description. Moreover, Blair (1987) demonstrated that the arrangement of excretory system in Neoctangium (with an elongate recurved bladder, lacking a rosette of diverticula proximal to the excretory pore, reception of excretory ducts at a single median point proximally; a ventral excretory pore) is highly dissimilar from that of Octangium spp and resurrected Neoctangium based upon. Hence, the genus today remains bispecific, with O. sagitta and O. hyphalum Blair, 1987, first described from the Pseudocaecum and large intestine of also the same host, C. Mydas from the western central pacific, off Queensland (Blair, 1987). In the following section, we discuss previous synonymies proposed within this genus.

Two species have been synonymised with the type species O. sagitta. The first is O. takanoi (Blair, 1987). Octangium takanoi was first described from the same host as O. sagitta, off Singapore, Western Central Pacific (Kobayashi, 1921). Blair (1987) synonymised it with O. sagitta based on examining figures of Kobayashi (1921). Even though the eggs of O. takanoi and those of O. sagitta do indeed overlap (despite Kobayashi (1921) distinguishing it from its congeners by eggs size), O. takanoi can be easily distinguished from O. sagitta by being twice smaller (5500 ×1200 vs. 4231–10250 × 1472–2424), by the shape of the oral sucker (rectangular vs. rounded) and especially by the extent of vitellaria, with their anterior end situated at the level of the anterior margin of the testes in O. takanoi (Fig. 7A), while they extend to the intestinal bifurcation in O. sagitta (Fig. 7G, H). Additionally, they can be easily distinguished by more anterior placing of the testes in O. takanoi (both testes are in the anterior half in O. takanoi. We refute thus the synonymy between the two species, and we reinstate O. takanoi as a valid species.

Octangium takanoi was reported by Mehrotra (1973) in an abstract communicated by N. K. Gupta from India without giving any additional information (Blair, 1987). Subsequently, Tandon and Gupta (1981) provided a redescription and morphometrical data of O. takanoi from the intestine of C. mydas from the Gulf of India, Eastern Indian Ocean. The microscaphidiids reported by Tandon and Gupta (1981) (that we refer to in Table 4 as O. takanoi sensu Tandon and Gupta (1981)) are larger than O. takanoi and have larger eggs. Most importantly, in O. takanoi the vitellarium extends to the anterior margin of the anterior testis while in O. takanoi sensu Tandon and Gupta (1981), the vitellarium extends about the middle of the posterior testis (Fig. 7B) and the two populations can be easily distinguished by the more anterior placement of tests in O. takanoi. Hence, the microscaphidiids reported by Tandon and Gupta (1981) are not O. takanoi. We note that O. takanoi sensu Tandon and Gupta (1981) resembles O. hyphalum, in which the vitellarium’s anterior limit near the point of contact between testes (Fig. 7C) and the measurements of different body parts and of eggs overlap. Hence, as previously suggested by Blair (1987), the two populations are highly likely conspecific.

Table 4

Comparative measurements for *Octangium hasta* Looss, 1902. * Diameter.¹
Host	Chelonia mydas	Chelonia mydas	Chelonia mydas	Chelonia mydas
Habitat	Large intestine	ED	ED	?
Locality	Egypt, EM.	Cairo, Egypt, EM.	Cairo, Egypt, EM.	Cairo, Egypt, EM.
Number of specimens	NA	30 adults	12 juveniles	34
Reference	(Looss, 1902).	Present study	Present study	Looss’s (1902) type material from NHR collections (Blair, 1987)
Body length	3–3300, max 4000	2953 (2148–3950, n = 30)	2187 (1569–2942, n = 12)	2940 (2150–3880)
Body width	Up to 900	789 (595–1128, n = 30)	577 (412–754, n = 12)	830 (630–1150)
Oral sucker length		187 (124–251, n = 27)	162 (135–198, n = 12)	190 (150–240)
Oral sucker width		199 (117–261, n = 27)	158 (128–195, n = 12)	200 (150–270)
Oesophagus length		758 (509–1049, n = 30)	595 (445–717, n = 12)	770 (540–1000)
Distance anterior end- Intestinal bifurcation		977 (709–1278, n = 30)	758 (555–904, n = 12)
Distance anterior testes- intestinal bifurcation		439 (302–579, n = 30)	344 (205–502, n = 11)	370 (200–490)
Anterior testis length		236 (147–470, n = 28)	109 (35–227, n = 11)	230 (130–380)
Anterior testis width		157 (107–281, n = 28)	73 (20–154, n = 11)	200 (110–380)
Posterior testis length		246 (129–500, n = 26)	115 (32–223, n = 11)	230 (110–380)
Posterior testis width		156 (99–270, n = 26)	71 (24–145, n = 11)	190 (100–370)
Ovary length		134 (94–203, n = 8)	72 (66–78, n = 2)	100 (60–150)
Ovary width		105 (67–249, n = 8	57 (56–57, n = 2)	80 (60–110)
Number of excretory diverticula
Eggs length	84	78 (68–85, n = 25)		71–87
Eggs width	50	52 (41–62, n = 25)		48–58
Abbreviations: NA, not available. E.M., Eastern Mediterranean.

Table 5

Comparative measurements for *Octangium* spp. * Diameter.¹
Species	Octangium sagitta	O. hasta	O. hyphalum Blair, 1987	O. takanoi Kobayashi, 1921	O. takanoi sensu Tandon and Gupta (1981)
Host	Chelonia mydas
Habitat	ED	ED	Pseudocaecum, large intestine	Intestine	Intestine
Locality	Cairo, Egypt, E. M.	Cairo, Egypt, EM.	Queensland	Singapore, Western Central Pacific	India, Eastern Indian Ocean
Number of specimens	13 adults	30 adults	19	NA	NA
Reference	Present study	Present study	(Blair, 1987)	(Kobayashi, 1921)	Tandon and Gupta (1981)
Body length	7061 (4231–10250)	2953 (2148–3950)	3040–6520 (4580)	5500	5160–7800
Body width	1916 (1472–2424)	789 (595–1128)	1100–2500 (1710)	1200	1240–1980
Oral sucker length	313 (248–360)	187 (124–251)	220–460 (320)		290–400
Oral sucker width	432 (337–518)	199 (117–261)	300–530 (380)		170–340
Oesophagus length		758 (509–1049)	620–1280 (910)		1420–2060
Number of excretory diverticula					6
Distance anterior end- Intestinal bifurcation	1867 (1249–2411)	977 (709–1278)
Distance anterior testes- intestinal bifurcation	1112 (417–1773)	439 (302–579)	80–280 (170)
Anterior testis length	860 (45 − 1174)	236 (147–470)	300–940 (540)		400–890
Anterior testis width	663 (186–1068)	157 (107–281)	280–1040 (530)		360–990
Posterior testis length	885 (471–1243)	246 (129–500)	280–1030 (540)		410–870
Posterior testis width	649 (194–1043)	156 (99–270)	240–980 (510)		330–990
Ovary length	373 (250–521)	134 (94–203)	120–480 (240)		150–230
Ovary width	293 (230–454)	105 (67–249)	110–300 (200)		90–250
Eggs length	77 (70–81)	78 (68–85)	67–79	70–75	58–78
Eggs width	51 (42–56)	52 (41–62)	44–50	45	44–53

Another current ambiguity within this genus is related to the type species of the genus, O. sagitta with the related synonyms. Octangium sagitta was first described by Looss (1899) as Microscapha sagitta Looss, 1899, from the rectum of the Green sea turtle C. mydas. In his subsequent comments on the names previously suggested for some “Distomid” genera, Looss (1900) renamed the corresponding genus Microscapha Looss, 1899, Microscaphidium Looss, 1900 before subsequently placing Microscapha sagitta in the latest genus (Looss, 1901). It was at last placed in the genus Octangium Looss, 1902 (Looss, 1902). Blair (1987) synonymised O. sagitta with O. hasta, first described from the large intestine of the same host, C. mydas from the Eastern Mediterranean, off Egypt (Looss, 1902). In the current study, we compared O. sagitta to O. hasta, and the two species can be easily distinguished by measurements, with O. hasta being significantly smaller in all body measurements (see also (Fig. 8), where all adult specimens were drawn to the same scale). More importantly, O. sagitta and O. hasta can be easily distinguished by the shape of the oral sucker (Fig. 7G, H vs. D-F). Thus, we reinstate O. hasta as a valid species. We summarize the distribution of current valid species in Fig. 9 and we provide a diagnostic key to species of the genus.

Key to Species

Lateral vitelline fields with anterior limit close to fork of caeca, oral sucker rounded....................O. sagitta

Lateral vitelline fields with anterior limit close to fork of caeca, oral sucker rectangular.................O. hasta

Lateral vitelline fields with anterior limit beyond point of contact between testes .......................O. takanoi

Lateral vitelline fields with anterior limit near point of contact between testes ........................O. hyphalum

Acknowledgments

We thank Dr. Christopher Blanar, Professor of Biology from the Nova Southeastern University. U.S.A. and Dr. Whitney Preisser, Professor at Kennesaw State University, Georgia (KSU) for suggesting the methods to rehydrate specimens. Special thanks to Adrian Lenninger for kindly generating accession numbers. This research was funded by the Swedish Taxonomy Initiative, Artdatabanken, Swedish University of Agricultural Sciences within the scope of the project “Taxonomy and systematics of digenetic trematodes parasitising fishes of Sweden” (dha 2019.4.3–48).

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The authors declare no competing interests.

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“Reviving museum oddities: rehydrating Trematodes curiosities!” A successful method for rehydrating dried museum specimens for a revision of the genus Octangium (Trematoda), parasites of green sea turtle Chelonia mydas

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Abstract

Figures

1. Introduction

2. Material and methods

2.1. Rehydration of dried specimens

2.2. Staining and slides mounting

2.3. Examination of comparative material

3. Results

3.1. Redescription (Figs. 3, 4)

1.1. Redescription (Figs. 5, 6)

4. Discussion

2.1. Looss’s Trematodes collection

2.2. Rehydration of dried specimens

2.3. Systematics of Octangium spp.

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Acknowledgments

References

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