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Journal of Wildlife Diseases, 42(2), 2006, pp. 386-390
© Wildlife Disease Association  2006
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SHORT COMMUNICATION

The Flea, Megabothris abantis: An Invertebrate Host of Hepatozoon sp. and a Likely Definitive Host in Hepatozoon Infections of the Montane Vole, Microtus montanus

Raychel A. Watkins1, Suzanne E. Moshier1,3 and Aelita J. Pinter2

1 Department of Biology, University of Nebraska at Omaha, Omaha, Nebraska 68182-0040, USA;
2 Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana 70148, USA
3 Corresponding author (email: smoshier{at}mail.unomaha.edu)

ABSTRACT:   In searching for an invertebrate host for Hepatozoon sp. infecting the montane vole (Microtus montanus), we collected fleas, ticks, and mites from live-trapped voles and searched squash preparations for Hepatozoon oocysts. From 1989 through 1996, we identified six species of fleas in Grand Teton National Park: Megabothris abantis, Megabothris asio megacolpus, Aetheca wagneri, Peromyscopsylla selenis, Peromyscopsylla. hesperomys, and Hystrichopsylla dippiei dippiei. We found Hepatozoon oocysts only in M. abantis; we found no oocysts in mites or ticks. We conclude that M. abantis is an invertebrate host of Hepatozoon sp. and is likely to be the definitive host for theHepatozoon spp. of M. montanus.
  Key words:  Definitive host, flea, Hepatozoon, invertebrate host, Megabothris abantis, Microtus montanus, oocyst, sporocyst.

Hepatozoon spp. parasitize many mammals, especially rodents (Smith, 1996). The life cycle includes a second host, the invertebrate or definitive host, which, depending upon species may be a tick, mite, louse, flea, mosquito, or reduviid bug (Krampitz, 1964; Smith, 1996). Although many mammals are known to be hosts for Hepatozoon sp., the corresponding invertebrate hosts are known for only a few. We sought to identify an invertebrate host for the Hepatozoon spp. parasitizing the montane vole, Microtus montanus. Because one of us (A.J.P.) is conducting a long-term study of the population dynamics of M. montanus (Pinter, 1986, 1988), we had the opportunity to collect invertebrate ectoparasites from montane voles live-trapped in Wyoming in Grand Teton National Park. Desser et al. (1995) found the search for the invertebrate host of Hepatozoon catesbianae "a slow and laborious process"; our search was also prolonged and labor intensive. We examined ectoparasites of M. montanus for five collection years before finding oocysts in the flea, Megabothris abantis.

We collected mites, ticks, lice, and fleas from M. montanus live-trapped in Grand Teton National Park, Wyoming, at approximately 43°50'N, 110°35'W. We squashed the arthropods and made wet mounts on 1 x 3–inch glass slides with phosphate-buffered saline to screen these ectoparasites for the presence of Hepatozoon oocysts. We used a compound microscope at 60x (4x objective lens and 15x ocular lens) and focused our examination on the abdominal region. Putative oocysts could be discerned, and specimens suspected of being infected were squashed, fixed in methanol, and stained with Wright–Giemsa stain. Squash preparations were examined further for the presence of Hepatozoon oocysts with a light microscope equipped with 15x oculars and a 100x oil-immersion objective lens. Fleas that were not squashed were preserved in 70% ethanol and sent to Robert E. Lewis at Iowa State University to be identified. He identified the two squashed fleas that contained oocysts from photographs as well as the other preserved fleas.

We drew blood from the left ventricle of the heart of euthanized voles with a 25-gauge needle on a heparinized tuberculin syringe and then prepared blood smears. Smears of femoral bone marrow and impression smears of lung, liver, and spleen were also prepared. The smears were fixed in methanol and stained with Wright–Giemsa stain. Each smear was examined for a minimum of 15 min with a compound microscope equipped with 15x oculars and a 100x oil objective.

We approached the search for an invertebrate host qualitatively at the start. Squashed fleas from our earliest attempts in 1988 were discarded because we were simply looking for a Hepatozoon-infected flea. We began keeping uninfected fleas to send to R.E. Lewis for identification after we noticed morphological differences among them (Table 1Go). From 1989 to 1994 we found two out of 23 M. abantis infected with Hepatozoon sp. Consequently, a rough approximation of the prevalence in M. abantis is 10%.


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  		TABLE 1. Identified fleas collected in Grand Teton National Park from the montane vole (Microtus montanus).

 
We found Hepatozoon oocysts in two M. abantis individuals collected in 1994. Figure 1Go shows several oocysts magnified 160x. The maximum oocyst diameter is 125 µm. This size fits neither category used by Smith and Desser (1997) in their phylogenetic analysis of the genus Hepatozoon. They characterized the maximum oocyst diameter of Hepatozoon spp. into one of two size ranges, <80 µm or >150 µm. Their study included two species, Hepatozoon erhardovae and Hepatozoon griseisciuri, that use mammalian intermediate hosts; both had oocysts with a maximum diameter >150 µm. Our data call into question the utility of this character.


Figure 1
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  		FIGURE 1. Squash preparation of Megabothris abantis infected with Hepatozoon sp. (Wright–Giemsa stain). Oocysts, magnification 160x, scale bar=50 µm.

 
In Figure 2Go, with magnification increased to 320x, sporocysts within the oocytes are evident. We found more than 100 sporocysts per oocyst, an observation that matches the characteristics reported by Smith and Desser (1997) for H. erhardovae and H. griseisciuri. At 1,600x (Fig. 3Go) sporozoites within the sporocysts are evident. The maximum sporocyst diameter is approximately 13 µm. This small size places the Hepatozoon spp. of M. abantis in the less-than-25-µm, rather than the greater-than-30-µm, category of Smith and Desser. It is the same category as H. erhardovae and H. griseisciuri. We saw four to six sporozoites per sporocyst. Hepatozoon erhardovae and H. griseisciuri also fell into the category with fewer than 16 sporozoites per sporocyst in the Smith and Desser study. The Hepatozoon spp. of M. abantis thus matches H. erhardovae and H. griseisciuri in three of the four characters (maximum oocyst diameter, number of sporocysts per oocyst, maximum sporocyst diameter, and number of sporozoites per sporocyst) we have evaluated.


Figure 2
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  		FIGURE 2. Squash preparation of Megabothris abantis infected with Hepatozoon sp. (Wright–Giemsa stain). Sporocysts in oocysts, magnification 320x, scale bar=25 µm.

 

Figure 3
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  		FIGURE 3. Squash preparation of Megabothris abantis infected with Hepatozoon sp. (Wright–Giemsa stain). Sporozoites within sporocysts, magnification 1,600x, scale bar=5 µm.

 
We collected six flea species (M. abantis, Megabothris asio megacolpus, Aetheca wagneri, Peromyscopsylla selenis, Peromyscopsylla hesperomys, and Hystrichopsylla dippiei dippiei) from 1989 to 1996. Fleas examined in 1988 were not retained for identification. We found oocysts only in M. abantis. This finding establishes M. abantis as an invertebrate host for Hepatozoon sp. Additionally, because M. abantis is the flea that we collected most frequently from M. montanus (Table 1Go), we propose that it is most likely the invertebrate host species for the Hepatozoon spp. of M. montanus. However, some caution is required because an infected flea may have acquired its infection from an animal other than the one on which it was found. A case in point is our finding of both infected fleas in 1994 on voles that were negative for Hepatozoon (Table 2Go). This situation could arise if the flea were infected from the host vole harboring an undetected infection or if it were infected from another animal.


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  		TABLE 2. Prevalence of Hepatozoon sp. in montane voles (Microtus montanus) in Grand Teton National Park.

 
We found that Hepatozoon sp. is a persistent endoparasite in populations of M. montanus in northwestern Wyoming. From 1988 to 1996, blood, bone marrow, lungs, liver, and spleen of 468 M. montanus were examined for infection with Hepatozoon sp. (Table 2Go). Thirty-nine of these voles were found to be infected (overall infection rate of 8.3%). The year with the highest, and atypical, rate of infection was 1989 with 19% infected. In two years, 1994 and 1995, no infections were found. In the other collection years the infection rates ranged from 8% to 12%. The variation in infection rate cannot be explained satisfactorily by the prevailing macroclimate. For example, both 1988 and 1994 were drought years, yet the infection rates, 12% and 0%, respectively, are quite different. We suspect that the microclimate experienced by fleas in the voles’ burrows may be more important than the macroclimate in determining the success of flea reproduction. Because flea larvae die when the relative humidity is below 45%, and they fail to mature at temperatures above 35 C (Powell, 1994), it is likely that burrows provide a more mesic microclimate than one would expect given the more xeric, drought-stricken meadows.

Our research establishes M. abantis as an invertebrate host for Hepatozoon sp. Because infected fleas were removed from M. montanus, it is also preliminary evidence that M. abantis transmits Hepatozoon to M. montanus. Transmission studies are necessary to confirm or refute this relationship. Completion of the life cycle of this Hepatozoon remains to be determined, and other vertebrate and invertebrate hosts may be involved in the life cycle.

Among rodents for which Hepatozoon infections have been reported, the invertebrate host is frequently unknown. Hepatozoon has been reported in other species of the genus Microtus, including Microtus agrestis (Healing, 1981), Microtus arvalis (Pawelczyk et al., 2004), Microtus oeconomus (Obayashi, 1971), Microtus californicus (Laakkonen et al., 2001), Microtus miurus (Laakkonen et al., 2002), and Microtus pennsylvanicus (O’Dell et al., 1991). Hepatozoon spp. have been reported in rodent species in other genera including Clethrionomys and Apodemus (Healing, 1981), Lemmus (Laakkonen, 2004), Liomys (Desser, 2000), Idiurus (Killick-Kendrick, 1984), Thomomys and Peromyscus (Moshier et al., 1994), and Sciurus (Clark, 1958). We are aware of one instance in which a flea is known to be the vector that transmits Hepatozoon. Frank (1977) reported that Megabothris turbidus transmits Hepatozoon erhardovae to a nonspecific host, the yellow-necked mouse (Apodemus flavicollis).

We are grateful to the University of Wyoming (UW) and the National Park Service (NPS) for the use of the UW-NPS Research Center in Grand Teton National Park and to Robert E. Lewis, Emeritus Professor of Entomology, Iowa State University, for identifying fleas. We are also grateful to two anonymous reviewers whose valuable comments improved this paper. This study was supported in part by a grant from UW.

LITERATURE CITED

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MOSHIER, S. E., W. D. O’DELL, R. A. WATKINS, AND A. J. PINTER. 1994. Parasites of small mammals in Grand Teton National Park: Babesia and Hepatozoon. In University of Wyoming–National Park Service Research Center 18th annual report, H. J. Harlow and M. Harlow (eds.). UW-NPS Research Center, Laramie, Wyoming, pp. 65–75.

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Received for publication 26 October 2004.




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