Helminth (Cestoda, Nematoda) and coccidian (Apicomplexa: Eimeriidae) parasites of the eastern small-footed myotis, Myotis leibii (Chiroptera: Vespertilionidae) from Arkansas, with a description of a new species of Eimeria

Abstract

During May and July 2016, 32 eastern small-footed myotis (Myotis leibii) were collected from five counties of northwestern Arkansas and their faeces examined for coccidian parasites. Four of 32 (13%) M. leibii harboured an eimerian that we describe here as new. Oocysts of Eimeria sassei sp. n. were ovoidal to ellipsoidal with a bi-layered wall and measured (length × width, L × W) 18.3 × 15.2 μm, with an L/W ratio of 1.2. A micropyle and oocyst residuum were absent but 1–2 polar granules were present. Sporocysts were ovoidal, 9.6 × 6.3 μm, with an L/W ratio of 1.5. A pronounced, button-like Stieda body was present but substieda and parastieda bodies were absent. A sporocyst residuum was present as distinct aligned or dispersed granules. One bat that we found dead was examined for helminth parasites. It harbored the tapeworm, Vampirolepis sp. and a nematode, Seuratum cancellatum. This is the first coccidian as well as the initial helminths reported from M. leibii. In addition, this is the seventh species of coccidian parasite documented from Arkansas bats.

Introduction

The eastern small-footed myotis, Myotis leibii Audubon and Bachman, 1842 is among the smallest and rarest vesper bats in eastern North America (Robbins et al. 1977). This bat is ranked S1 (critically imperiled) in Arkansas (NatureServe 2015). It ranges from Ontario, Canada and New England southward to Georgia and Alabama and westward into Oklahoma (Best and Jennings 1997; Reid 2006). In Arkansas, M. leibii has been reported from scattered localities in the Interior Highlands (Ouachitas and Ozarks) (Sealander and Heidt 1990).

Although information is available on the ecology and natural history of M. leibii (Best and Jennings 1997), nothing, to our knowledge, has been reported on any coccidian from this bat and there are no reports of cestodes or nematodes from the species. However, limited information is available on trematode parasites of M. leibii from Canada (Stafford 1905). Here, we provide a description of an eimerian from M. leibii from Arkansas as well as document, for the first time, two helminth parasites from this host in the state.

Materials and Methods

During May and July 2016, four juvenile and 28 adult M. leibii (13 males, 19 females [including nine pregnant, one lactating, and two post lactating) were taken by hand (with the use of a yardstick) from the crevices between sections of concrete guardrail on top of bridges in Carroll (n = 4), Franklin (n = 5), Madison (n = 6), Newton (n = 2) and Washington (n = 15) counties, Arkansas. Captured bats were allowed to defecate and fresh faecal pellets were placed in individual vials containing 2.5% (w/v) aqueous potassium dichromate (K₂Cr₂O₇). Samples were shipped to CTM and examined for coccidia by light microscopy after flotation in Sheather’s sugar solution (specific gravity = 1.30). Measurements were taken on 26 sporulated oocysts using a calibrated ocular micrometer and reported in micrometres (μm) with means followed by the ranges in parentheses; photographs were taken using Nomarski interference-contrast optics. Oocysts were ~78 days old when measured and photographed. Descriptions of oocysts and sporocysts follow guidelines of Wilber et al. (1998) as follows: oocyst length (L) and width (W), their ratios (L/W), micropyle (M), oocyst residuum (OR), polar granule(s) (PG), sporocyst length (L) and width (W), their ranges and ratio (L/W), sporocyst (SP), Stieda body (SB), substieda body (SSB), parastieda body (PSB), sporocyst residuum (SR), sporozoites (SZ) anterior (ARB) and posterior (PRB) refractile bodies, and nucleus (N).

A single adult female M. leibii collected by D. Blake Sasse in May 2016 from the St. Hwy 74 bridge in Washington County that died unexpectedly was frozen; it was shipped to CTM and examined for helminth parasites. A midventral incision was made from anus to throat, the gastrointestinal tract was removed, split longitudinally, its contents placed in a Petri dish containing 0.9% saline, and examined using a stereomicroscope. A tapeworm was fixed in 70% (v/v) ethanol, stained with acetocarmine, and mounted in Canada balsam. A nematode was fixed in ethanol and examined as a temporary mount in glycerol.

A host voucher (alcoholic specimen) was accessioned into the Mammal Collection of Henderson State University (HSU), Arkadelphia, Arkansas, USA as HSU 946. Photosyntypes of sporulated oocysts were accessioned into the Harold W. Manter Laboratory of Parasitology (HWML), Lincoln, Nebraska USA.

Results

Four of 32 (13%) M. leibii (all pregnant females) collected on 13 May 2016 from Franklin (one), Madison (two), and Washington (one) counties were found to be passing coccidian oocysts that we describe herein as new. In addition to the new coccidian, a single immature tapeworm, Vampirolepis sp. (HWML 102050) and a female nematode, Seuratum cancellatumChitwood, 1938 (HWML 98344) were found in the intestinal tract of an adult female M. leibii collected on 13 May 2016 from Washington County.

Eimeria sassei sp. nov. (Figs. 1–4)

Figs. 1–3.

Nomarski interference-contrast photomicrographs of oocysts of Eimeria sassei n. sp. 1. Oocyst showing oocyst wall (OW), polar granule (PG) and sporocyst residuum (SR). 2. Oocyst showing PG, Stieda body (SB) and sporocyst (SP). 3. Oocyst showing bi-layered OW and SB. Scale bars = 10 μm for all figures.

Fig. 4.

Composite line drawing of oocyst of Eimeria sassei n. sp. Scale bar = 10 μm.

Description of sporulated oocyst: Oocyst with 2 sporocysts; shape ovoidal to ellipsoidal; rough (mamillated) bi-layered wall, tan in colour, ~ 1.1 (1.0–1.2) thick, outer 0.8 (0.7–0.9) rough, inner 0.3 (0.3–0.4), smooth; L × W: 18.3 × 15.2 (16–21 × 14–17); L/W: 1.2 (1.1–1.3); M and OR absent; 1– (rarely) 2 PG, present.

Description of sporocyst and sporozoites: SP ovoidal; smooth uni–layered wall, ~ 0.5 thick; L × W: 9.6 × 6.2 (7–11 × 6–7); L/W: 1.5 (1.2–1.8); SB pronounced, button-like, SSB and PSB absent; SR: composed of distinct medium-sized coarse granules aligned along perimeter of SP or rarely dispersed; SZ: (not measured) sausage-shaped with spheroidal ARB and PRB; single N slightly posterior to midpoint.

Taxonomic summary

Type host: Eastern small-footed myotis, Myotis leibii Audubon and Bachman, 1842 (Mammalia: Chiroptera: Vespertilionidae), pregnant female band #7965. Collected and released on 13 May 2016. This bat was originally banded as a juvenile on 16 July 2015.

Type specimen: Photosyntype of sporulated oocyst deposited in the HWML as No. 102075.

Type locality: Madison County, Arkansas, USA (Eastling 429601, Northling 3964198).

Prevalence: In four of 32 (13%) of the type host. 0/4 (0%) Carroll County, 1/5 (20%) Franklin County, 2/6 (33%) Madison County, 0/2 (0%) Newton County, 1/15 (7%) Washington County.

Sporulation: Exogenous. All oocysts were passed unsporulated or partially sporulated and became fully sporulated within five days at ~23°C.

Prepatent and patent periods: Unknown.

Site of infection: Unknown, oocysts recovered from faeces.

Etymology: The specific epithet is given in honor of D. Blake Sasse, Nongame Mammal/Furbearer Program Leader, Arkansas Game and Fish Commission, for his longtime work on Arkansas mammals. Mr. Sasse also collected all of the bats examined in this study.

Remarks: Six coccidians have been described and/or reported previously from Arkansas bats (Table 1): Eimeria catronensis Scott and Duszynski from northern long-eared myotis, Myotis septentrionalis Trouessart, 1897 (McAllister et al. 2004); Eimeria dowleri McAllister and Upton from red bats, Lasiurus borealis Müller, 1776 (McAllister and Upton 2009); Eimeria heidti McAllister, Burt, Seville and Robison from tri-colored bats, Perimyotis subflavus F. Cuvier, 1832 (McAllister et al. 2011); Eimeria macyi Wheat from P. subflavus (McAllister et al. 2001); Eimeria mcdanieli McAllister, Seville, Arlen and Connior from P. subflavus (McAllister et al. 2014); and E. sealanderi McAllister and Upton from L. borealis (McAllister and Upton 2009) (Table 2). Nearly all of these possess oocysts and sporocysts that are much larger than the new species and/or they possess an M (E. catronensis), an OR (E. sealanderi) or a SSB (E. macyi), all of which are absent in E. sassei (Table 2).

Table 1.

Coccidians reported from Arkansas bats.

Coccidian	Host	Prevalence*	Reference
Eimeria catronensis	Myotis septentrionalis	4/20 (20%)	McAllister et al. (2004)
E. dowleri	Lasiurus borealis	2/3 (67%)	McAllister and Upton (2009)
E. heidti	Perimyotis subflavus	2/20 (10%)	McAllister et al. (2011)
		2/11 (18%)	McAllister et al. (2014)
E. macyi	P. subflavus	2/5 (20%)	McAllister et al. (2001)
E. mcdanieli	P. subflavus	2/11 (18%)	McAllister et al. (2014)
E. sassei	Myotis leibii	4/32 (13%)	This study
E. sealanderi	L. borealis	2/3 (67%)	McAllister and Upton (2009)

^*Prevalence = number infected/number examined.

Table 2.

Comparison of the sporulated oocysts of seven eimerians reported from Arkansas bats.

Eimeria spp.	Type host and other host Localities	Oöcyst shape, size, features*†	Sporocyst shape, size, features*†	References
E. catronensis	Myotis lucifugus	Ovoidal	Football-shaped	Scott and Duszynski, 1997
	New Mexico, USA	22.2 × 14.8; L/W 1.5	9.1 × 6.6; L/W 1.2
		18–25 × 14–17	8–11 × 5–7
	Myotis septentrionalis	M, PG: both +	SB, SR: both +	McAllister et al. 2004
	Arkansas, USA	OR: −	SSB, PSB: both −
E. dowleri	Lasiurus borealis	Subspheroidal to ellipsoidal	Ovoidal	McAllister and Upton, 2009
	Arkansas, USA	24.7 × 22.0; L/W 1.1	13.4.× 9.2; L/W 1.5
		23–26 × 20–23	12–14 × 8–9
		M, OR: both −	SB, SR: both +
		PG: +	SSB +, PSB: −
E. heidti	Perimyotis subflavus	Ovoidal to ellipsoidal	Ovoidal	McAllister et al. 2011
	Arkansas, USA	26.1 × 20.5; L/W 1.3	13.0 × 8.8; L/W 1.6
		23–31 × 18–23	11–15 × 7–13
		M, OR: both −	SB, SR: both +
		PG: +	SSB, PSB: both −
E. macyi	P. subflavus	Subspheroidal to broadly ellipsoidal	Ovoidal	Wheat, 1975
	Alabama, USA	19.0 × 17.6; L/W 1.1	11.0 × 7.0; L/W 1.6
		16–21 × 15–19	10–12 × 6–8	McAllister et al. 2001
	Arkansas, USA	M, OR: both −	SB, SR: both +
		PG: +	SSB +, PSB: −
E. mcdanieli	P. subflavus	Ellipsoidal to elongate	Ovoidal	McAllister et al., 2014
	Arkansas, USA	28.3 × 17.9; L/W 1.6	12.6 × 8.3; L/W 1.5
		25–30 × 16–20	11–14 × 8–9
		M, OR: both −	SB, SR: both +
		PG: +	SSB +; PSB: −
E. sassei	Myotis leibii	Ovoidal to ellipsoidal	Ovoidal	This study
	Arkansas, USA	18.3 × 15.2; L/W 1.2	9.6 × 6.2; L/W 1.5
		16–21 × 14–17	711 × 6–7
		M, OR:both −	SB, SR: both +
		PG: +	SSB, PSB: both −
E. sealanderi	L. borealis	Subspheroidal to ellipsoidal	Ovoidal	McAllister and Upton, 2009
	Arkansas, USA	16.7 × 14.4; L/W 1.1	8.9.× 5.7; L/W
		15–18 × 13–16	8–10 × 5–6
		M: −	SB, SR: both +
		OR, PG: both +	SSB +, PSB: −

^*Measurements in μm.

^†Descriptions of oöcysts and sporocysts follow guidelines of Wilber et al. (1998) as follows: oöcyst length (L) and width (W), their ranges and ratios (L/W), micropyle (M), oöcyst residuum (OR), polar granule(s) (PG), sporocyst (SP) length (L) and width (W), their ratio (L/W), Stieda body (SB), sub-Stieda body (SSB), para-Stieda body (PSB), and sporocyst residuum (SR).

Eimeria tumlisoni McAllister, Seville and Roehrs from M. septentrionalis from Oklahoma is most similar to E. sassei (McAllister et al. 2012). However, there are several morphological and mensural differences that distinguish E. tumlisoni from E. sassei as follows: (i) oocysts of E. tumlisoni are subspheroidal vs. ovoidal to ellipsoidal in E. sassei, (ii) the L/W ratio of E. tumlisoni sporocysts are greater (average 1.8) than in E. sassei (average 1.5), (iii) E. tumlisoni SR are dispersed vs. distinctly aligned along the sporocyst in E. sassei, and (iv) the E. tumlisoni oocyst wall is much thicker (1.3–1.7 vs. 1.0–1.2) than the new species (McAllister et al. 2012). All of the other coccidians reported from North American bats of the genus Myotis are different than E. sassei (see Duszynski 2002).

Discussion

Tapeworms of the genus Vampirolepis are commonly reported from bats in North America (Rausch 1975; Vaucher 1992). This tapeworm was previously reported from Rafinesque’s big-eared bat, Corynorhinus rafinesquii Lesson, 1827 from Arkansas (McAllister et al. 2005). The ascarid, S. cancellatum was originally described from the Mexican greater funnel-eared bat, Natalus mexicanus Miller, 1902 from the Yucatán, México (Chitwood 1938). This parasite shows little host specificity as it has been reported from several other bats of the genera Antrozous, Corynorhinus, Eptesicus, Eumops, Myotis, Parastrellus, and Tadarida from Texas, USA (Specian and Ubelaker 1976). In Arkansas, McAllister et al. (2004) previously reported S. cancellatum from M. septentrionalis. These two helminths are the initial cestode and nematode reported from M. leibii and the second time they have been reported from an Arkansas bat. There is limited information on trematode parasites of M. leibii as Stafford (1905) reported two species of Lecithodendrium and Plagiorchis vespertilionis (Müller) from specimens collected from Canada.

Compared to rodents, the largest class of mammals, comparatively little is known about the coccidians of second largest class of mammals, the bats of the world (Duszynski 2002). We document the first coccidian parasite reported from M. leibii. A list of the coccidians reported from Arkansas bats are given in Table 1. Of the 16 species of bats that occur in the state (Sealander and Heidt 1990), only four species (25%) have been reported to harbor coccidians. Of the other 12 species from Arkansas that have not yet been reported to be passing coccidia in the state, the following have been reported to be infected in other localities, including big brown bats, Eptesicus fuscus (Palisot de Beauvois, 1796), hoary bats, Lasiurus cinereus (Palisot de Beauvois, 1796), and little brown myotis, Myotis lucifugus (LeConte, 1831) (Duszynski 2002). However, other bats in the state have been surveyed including Brazilian free-tailed bats, Tadarida brasiliensis (I. Geoffroy, 1824) and C. rafinesquii but were not found to be passing coccidians when examined (McAllister et al. 2005, 2006).

It is interesting to note that of the infected bats (n = 4), all were collected on the same date in May 2016. However, none of the 21 bats collected from the same sites over three days in mid-July 2016 were found to be passing oocysts. This may suggest that collections of bat fecal samples should be made during different months (and seasons) throughout the year to increase the possibility of finding individuals infected with coccidia.

The current study involved hosts collected from non-cave habitat due to a moratorium on collecting bats from caves and mines of the state to prevent the spread of devastating white-nose syndrome (caused by the fungus Pseudogymnoascus destructans, Frick et al. 2010), recently confirmed in Arkansas (https://www.whitenosesyndrome.org/news/white-nose-syndrome-confirmed-arkansas). Indeed, additional studies, similar the current one, can only occur with the assistance of state and federal officials who are able to legally and properly obtain fecal specimens from bats and pass them on to researchers. We may then, and only then, be able to extend our knowledge of the coccidia of many species of bats that, to date, have not yet been reported to harbor these parasites.