Azygiid Parasites Of North American Endemic Pleurocerids And Centrarchids: Revision Of Leuceruthrus Marshall And Gilbert, 1905 (Digenea: Azygiidae), Description Of Two New Species, And Phylogenetic Analysis Author Bullard, Matthew R. Womble and Stephen A. text Journal of Parasitology 2022 2022-08-18 108 4 374 394 http://dx.doi.org/10.1645/22-36 journal article 58023 10.1645/22-36 7336dbb8-69cd-4d08-a46b-9279b3abaafd 1937-2345 7753943 Leuceruthrus blaisei n. sp. ( Figs. 15 17 ; Table II) Diagnosis of cercaria (based on light microscopy of 3 stained, whole-mounted cercariae sourced from crushed snails) ( Figs. 15 17 ): Cercaria 2,060 2,590 (2,337, 3) long, comprised of a tail stem and paired furcae ( Fig. 15 ). Tail stem lacking pigmentation, 1,970 2,500 (2,237, 3) long or 95 97% (96%, 3) of cercariae length, maximum width 420 480 (447, 3) or 4.7 5.6 (5, 3) X longer than wide, comprised of an anterior and posterior region ( Fig. 15 ) bearing many unarmed papillae like tegumental projections ( Figs. 15, 16 ); anterior tail stem region (ATS) globular, tapering slightly anteriorly, widest medially, heavily tapering posteriorly, 990 1,320 (1,117, 3) long or 44 51% (48%, 3) of cercariae length, maximum width same as reported for tail stem, containing distome; posterior tail stem region (PTS) dorsoventrally compressed, 980 1,200 (1,120, 3) long or 46 51% (48%, 3) of cercariae length, widest medially, 250 360 (300, 3) wide, tapering anteriorly and posteriorly. Furcae lacking pigmentation, paired, small, oblong, dorsoventrally compressed ( Fig. 15 ); dorsal furca, 90 100 (93, 3) or 3 4% (4%, 3) of cercariae length, 70 140 (100, 3) wide; ventral furca 70 90 (80, 3) or 3 4% (3%, 3) of cercariae length, 70 130 (93, 3) wide ( Fig. 17 ). Tail cavity opening at anteromedial end of cercaria, directing anteriad. Tail stem projections irregularly distributed throughout tail stem, densely distributed near tail cavity opening, and posterior end of posterior tail stem. Tail stem spines not evident. Excretory system with 1 primary excretory canal, extending posteriad along medial axis of posterior tail stem, bifurcating at synthesis of furcae, extending independently through each furcae, opening via excretory pore at the distal end of each furcae. Table III. ITS2 sequence comparisons used herein. Percent similarity (upper) and nucleotide differences (bottom) (355 aligned length of fragment).*

L. blaisei n. sp. ,	L. blaisei n. sp. ,	L. micropteri ,	L. micropteri , adult,	L. ksepkai n. sp. ,
Elimia sp. 2 ,	Elimia sp. 2 ,	adult, M. salmoides ,	M. salmoides ,	E. floridensis ,
Simmons Creek, AL	Simmons Creek, AL	Wheeler Reservoir, AL	Wheeler Reservoir, AL	Holmes Creek, FL
Leuceruthrus blaisei n. sp. ,	100	96.7	96.7	99.0
Elimia sp. 2 , Simmons
Creek, Alabama
Leuceruthrus blaisei n. sp. ,	0	96.7	96.7	99.0
Elimia sp. 2 , Simmons
Creek, AL
Leuceruthrus micropteri , adult,	10	10	100	97.0
Micropterus salmoides ,
Wheeler Reservoir, AL
Leuceruthrus micropteri , adult,	10	10	0	97.0
Micropterus salmoides ,
Wheeler Reservoir, AL
Leuceruthrus ksepkai n. sp.,	3	3	9	9
Elimia floridensis , Holmes
Creek, FL
Leuceruthrus cf. ksepkai ,	4	4	10	10	3
Micropterus salmoides ,
Holmes Creek, FL
Leuceruthrus cf. ksepkai ,	5	5	11	11	4
Micropterus salmoides ,
Holmes Creek, FL
Leuceruthrus cf.	9	9	5	5	8
stephanocauda , Elimia cf.
carinifera , Big Canoe Creek,
AL
Proterometra epholkos	24	24	26	26	23
Proterometra ariasae	25	25	28	28	25
Azygia longa	26	26	26	26	25
Bivesicula unexpecta	113	113	111	111	112

*AL, Alabama; FL, Florida Body of distome (¼cercarial body) ( Fig. 15 ) oblong, 790 990 (893, 3) long or 38% (3) of cercaria length 340 440 (380, 3) wide at level oral sucker or 2.3 2.5 (2.4, 3) X longer than wider, contained within anterior tail stem, anterior margin 100 180 (150, 3) from tail cavity opening; forebody 370 470 (433, 3) long or 47 51% (48%, 3) of overall distome length; hindbody 240 310 (273, 3) long or 30 31% (31%, 3) of overall distome length, 59 66% (63%, 3) of forebody length; tegument unarmed. Excretory system not evident. Nervous system not evident. Oral sucker subterminal, anterior end 40 50 (43, 3) from anterior end of distome, posterior end 510 605 (572, 3) from posterior end of distome, 230 285 (258, 3) long or 29% (3) of distome length, 260 310 (277, 3) wide or 70 76% (73%, 3) of distome width, posterior margin 90 175 (138, 3) from anterior margin of ventral sucker ( Fig. 15 ). Ventral sucker nearly entirely positioned in posterior half of distome, 180 215 (192, 3) or 20 23% (22%, 3) of distome length, 200 250 (222, 3) or 57 60% (58%, 3) of distome width, 69 78% (74%, 3) of oral sucker length 77 83% (80%, 3) of oral sucker width ( Fig. 15 ). Pharynx ovoid, dorsal to oral sucker, 85 100 (92, 3) long, 60 75 (68, 3) wide ( Fig. 15 ). Esophagus extending posteriad from mouth before bifurcating posterior to pharynx, esophageal branches arching posterolaterad before joining with intestinal ceca. Intestinal ceca confluent with esophageal branches, appearing inverse U shaped inclusive of esophageal branches, comprising paired dextral and sinistral ceca ( Fig. 15 ); dextral cecum 655 865 (768, 3) long or 83 87% (85%, 3) of distome length, laterad/ascending cecum length 105 175 (132, 3), descending cecum length 550 690 (637, 3), prececal space 250 330 (292, 3) or 32 33% (33%, 3) of body length from anterior end of distome, postcecal space 50 55 (53, 3) from posterior end of distome; sinistral cecum 635 785 (720, 3) or 79 82% (81%, 3) of distome length, laterad/ascending cecum length 90 115 (102, 3), descending cecum length 545 670 (618, 3), prececal space, 235 295 (265, 3) or 29% (3) of distome length from anterior end of distome, postcecal space 55 85 (68, 3) from posterior end of distome. Testes 2 in number, abreast, round to oval ( Fig. 15 ), preovarian, near posterior margin of ventral sucker; dextral testis 40 70 (53, 3) long, 30 50 (38, 3) wide; sinistral testis 45 75 (55, 3) long, 30 55 (40, 3) wide, pretesticular space, 490 610 (563, 3) from anterior end of distome or 60 68% (63%, 3) of total body length, posttesticular space 240 270 (250, 3) from posterior end of distome or 27 30% (28%, 3) of total body length. Prostatic sac immediately anterior of ventral sucker. Genital atrium circular in ventral view, 35 45 (28, 2) in diameter. Genital pore anterior to ventral sucker, medial, 835 960 (877, 5) of body length from anterior end of body or at 52 59% (55%, 5) of body length. Features of terminal male genitalia (i.e., seminal vesicle, pars prostatica, ejaculatory duct, sinus organ) appearing as anlagen, difficult to discern. Table III. Extended.

L . cf. ksepkai , M. salmoides ,	L . cf. ksepkai , M. salmoides ,	L . cf. stephanocauda , E . cf. carinifera ,
Holmes Creek, FL	Holmes Creek, FL	Big Canoe Creek, AL	P. epholkos	P. ariasae	A. longa	B. unexpecta
98.7	98.4	97.0	92.3	91.7	91.4	64.6
98.7	98.4	97.0	92.3	91.7	91.4	64.6
96.7	96.4	98.4	91.6	90.8	91.4	65.2
96.7	96.4	98.4	91.6	90.8	91.4	65.2
99.0	98.7	97.4	92.6	91.7	91.8	64.9
99.7	97.0	92.3	91.5	91.5	64.8
1	96.7	92.0	91.1	91.2	64.8
9	10	91.3	90.4	91.1	64.6
24	25	27	92.6	92.5	65.2
26	27	29	23	90.5	65.5
26	27	27	23	29	65.3
113	113	113	111	110	111

Ovary medial, intercecal, posterior to testes, near posterior end of body or 150 200 (173, 3) of body length from posterior end of distome, 55 65 (60, 3) long or 1.4 1.8 (1.6, 3) X longer than wide, 30 45 (38, 3) wide. Fine features of terminal female genitalia (i.e., oviduct, Laurer’s canal, ovovitelline duct, oötype, and Mehlis’ gland) difficult to discern. Uterus 155 250 (210, 3) long or 20 25% (23%, 2) of body length, narrow, sinuous, extending in a near straight line from ovary, passing between testes, difficult to discern distally. Metraterm not evident. Vitellarium not developed in distome. Taxonomic summary for Leuceruthrus blaisei n. sp. Type host: Elimia sp. 2 . Type locality: Simmons Creek ( 31°20 ′ 59 ′′ N , 86°44 ′ 10 ′′ W ), Alabama . Specimens and sequences deposited: Holotype (cercaria, USNM 1593592 ), paratypes (cercariae, USNM 1593593 , 1593594 ); ITS2 (cercariae from E . sp. 2, GenBank ON 877228, ON877229 ). Prevalence of infection: 11 of 210 (5%) specimens of Elimia sp. 2 from Simmons Creek were infected. ZooBank registration: urn:lsid:zoobank.org:act: 8CAC2CB1- 76BE-468D-B512-7A9E49FD4459 . Etymology: The specific epithet blaisei honors M.R.W.’s friend, Blaise Sheridan (Professional Staff, U.S. Senate Appropriations Committee, Subcommittee on Commerce, Justice, Science, and Related Agencies), for his steadfast devotion to support and expand opportunities for natural sciences research and is in gratitude for his many years of support as a colleague. Taxonomic remarks on Leuceruthrus blaisei n. sp. The cercaria of the new species differs from that of its congeners by the combination of having proportionally small furcae (>tail stem maximum width) with a nipple-like distal projection and numerous minute projections on the tail stem surface. No other described cercaria of Leuceruthrus has this combination of features ( Faust, 1921 ; Horsfall, 1934 ; Smith, 1935 ; present study). Although our specimens came from crushed snails, the distome of each cercaria was withdrawn and fully developed ( Fig. 16 ); indicating that the cercariae were to be shed soon. Results of ITS2 nucleotide comparison and phylogenetic analysis As indicated above and confirming our morphological assessments, the 2 ITS2 sequences from cercariae of L. blaisei shed from Elimia sp. 2 in Simmons Creek were identical. The 2 adults of L. micropteri from largemouth bass in Wheeler Reservoir also were identical to each other. The ITS2 sequences from L. blaisei differed from those of L. micropteri , L. ksepkai , L . cf. ksepkai (2 isolates both from largemouth bass), and L . cf. stephanocauda by 3.3% (10 nucleotides), 1.0% (3 nucleotides), 1.3% and 1.6% (4 and 5 nucleotides), and 3.0% (9 nucleotides), respectively (Table III). We sequenced a cercaria of L. kspekai and 2 adults of L . cf. ksepkai . The 2 adult sequences of L . cf. ksepkai differed from that of the cercaria by 3 or 4 nucleotides, respectively. Our phylogenetic analysis ( Fig. 18 ) included all of our new sequences plus all publicly available ITS2 sequences for Leuceruthrus spp. and Proteromera spp. and recovered Leuceruthrus as monophyletic. The phylogenetic analysis recovered L. ksepkai and L . cf. ksepkai in a polytomy. Leuceruthrus blaisei was monophyletic (identical sequences) and L. micropteri and L . cf. stephanocauda were recovered as sister taxa. Proterometra was recovered as paraphyletic, with low nodal support, and A. longa , which infects an amnicolid (not a pleurocerid), Amnicola limosus ( Sillman, 1962 ) (Caenogastropoda: Rissooidea: Amnicolidae ) ( Fig. 18 ). Gibson (2002) accepted existing subfamilies and assigned Azygia , Otodistomum , and Proterometra to Azygiinae and Leuceruthrus to Leuceruthrinae Goldberger, 1911 . Our preliminary analysis supported the monophyly of Leuceruthrinae , as evidenced by the recovery of monophyletic Leuceruthrus sister to all other azygiids. Our tree lacks the taxon sampling to test monophyly or interrelationships of another azygiid genus (and it was not the objective of our present study to do so) but our preliminary result seemingly rejects monophyly of Azygiinae Lühe, 1909. Additional species of Proterometra and Leuceruthrus must be sequenced to test these relationships adequately. Also important is to describe and sequence the ITS2 of Otodistomum spp. in addition to using 28S sequences.