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Rare case of Microsporidia co-infection in the grasshopper Dichroplus elongatus (Orthoptera: Acrididae: Melanoplinae)
Carlos E. Lange12 and Maria Marta Cigliano13
1 Centro de Estudios Parasitologicosy de Vectores (CEPAVE), CCTLa Plata CONICET - Universidad Nacional de La Plata (UNLP), Argentina
2 Comision de Investigaciones Cientificas de la Provincia de Buenos Aires (CICPBA), Argentina
3 Museo de La Plata, Division Entomologia, Facultad de Ciencias Naturales, Universidad Nacional de La Plata (UNLP), Argentina
| Submitted October 24, 2018 | Accepted November 23, 2018 |
Summary
Two Microsporidia species are known to parasitize Dichroplus elongatus in the Pampas region of Argentina, allochthonous Paranosema locustae, a generalist pathogen of the adipose tissue of grasshoppers with efficient horizontal and vertical transmission routes, and autochthonous Liebermannia dichropluse, a D. elongatus-specific pathogen of the Malpighian tubules with effective transovarial transmission but unknown horizontal transfer. Long-term monitoring of grasshopper communities for detection of microsporidioses revealed that it is not often that these two pathogens co-exist in populations of D. elongatus, even in different individuals. We report the unusual detection for the first time of a co-infection by P. locustae and L. dichroplusae in D. elongatus at a site in the southern Pampas. Given the own traits that characterize both pathogens we presume that the most likely scenario for the occurrence of the observed mixed infection is that the affected D. elongatus individual was already infected by L. dichroplusae when it contracted infection by P. locustae. Unfortunately, our attempts to induce infections with L. dichroplusae in the laboratory through experimental inoculations have not produced positive results, preventing efforts towards testing our presumption through laboratory bioassays. However, Microsporidia natural mixed infections appear to be so rare that we believe this case is worth reporting.
Key words: Liebermannia dichroplusae, Paranosema locustae, mixed infection, transmission
Introduction
The univoltine and polyphagous melanopline grasshopper Dichroplus elongatus is widely distributed
in southern South America. It inhabits all ofArgen-tina except the island of Tierra del Fuego at the southernmost tip of the country, Uruguay, much of Chile, and southern Brazil (Cigliano et al., 2014).
doi:10.21685/1680-0826-2019-13-1-2 © 2019 The Author(s)
Protistology © 2019 Protozoological Society Affiliated with RAS
It is particularly common in natural and disrupted grasslands, pastures, and crops in the Pampas region (provinces of Buenos Aires, eastern La Pampa, and southern Cordoba and Santa Fe) where it readily becomes a pest when outbreaks develop (Cigliano et al., 2014; Carbonell et al., 2017). Two species of Microsporidia are known to parasitize D. elongatus in the Pampas, allochthonous Paranosema locustae and autochthonous Liebermannia dichropluse (Sokolova et al., 2007; Bardi et al., 2012). Paranosema locustae became naturalized in grasshopper communities after introductions for biocontrol purposes were done between years 1978-1982 (Lange and Cigliano, 2005). Liebermannia dichroplusae appears to be endemic to the Pampas since it has never been detected anywhere else outside that region in spite of years of surveys in Argentina. While P. locustae infects primarily the adipocytes of the host’s fat body tissue, exhibits an extremely broad host range among grasshoppers (at least 124 species are known to be susceptible worldwide; Lange, 2005, 2010, and unpublished observations; Phithalsoun and Zhang, 2018), has both horizontal and vertical transmission (Solter et al., 2012), and causes epizootics (Lange and Cigliano, 2005), L. dichroplusae develops in the Malpighian tubules of D. elongatus, appears to be host-specific, horizontal transmission has never been achieved, and occurs enzootically (Lange, 1987, 1997, 2003). After nearly thirty years of monitoring only a few sampling sites in the Pampas revealed the coexistence of P. locustae and L. dichroplusae albeit in different individuals of D. elongatus and not at the same time. Hereafter the first detection of a mixed infection (i.e. co-infection) by these two microsporidia is reported.
Material and methods
In early March 2017 a sample of adults of D. elongatus (n = 87) was collected with entomological nets in a disrupted grassland close to the intersection of highways 76 and 85 (37°46’33.63”S, 61°42’53.52”W, San Eloy locality, approximately halfway between the towns of Coronel Suarez and Coronel Pringles) in southern Buenos Aires province. Grasshoppers were immediately taken to the laboratory and frozen at -32 °C until processing. Upon thawing, small samples of tissues and organs were examined as wet mounts in one-quarter-strength Ringer’s solution (Poinar and Thomas, 1984) under phase contrast
microscopy (400*, 1000*) after careful ventral longitudinal dissection of each individual (Lange and Henry, 1996). After examination by dissection each grasshopper was homogenized whole in 5 ml of double distilled water, filtered through cheesecloth, and aliquots of resulting homogenates were further scrutinized under the compound microscope (Plischuk et al., 2013). Intensity of infections was estimated by spore counts per individual using an improved Neubauer haemocytometer (Undeen and Vavra, 1997). P. locustae and L. dichroplusae are readily distinguished by size and shape of spores, type of development stages, tissue-organ tropism, and internal pathological changes (Lange, 1987, 2003; Henry and Oma, 1981).
Although transovarial transmission is a well-established trait of L. dichroplusae (Lange, 1997), we still believe that some contribution through horizontal transfer should occur because there is no assistance from the male parent and infections do not seem to impart a selective advantage to the host but are indeed detrimental (Lange, 1987), conditions which are considered as requirements for the maintenance of the pathogen through vertical transmission alone (Fine, 1984). Accordingly, in order to induce horizontally transmitted infections we performed over the years additional oral experimental inoculations to those already reported (Lange, 1989) on all postembryonic developmental stages of D. elongatus (nymphal instars I to V and young, one-week old adults) following protocols successfully employed previously with other pathogens (Habtewold et al., 1995; Lange and Wittenstein, 1998). The source ofL. dichroplusae spores used was infections at the type locality (Brandsen) and other localities in Buenos Aires province (Lange, 2003). Briefly, a total of 291 individuals of D. elongatus reared at the Center for Parasitological Studies and Vectors (CEPAVE) as described by Mariottini et al. (2011) were individually placed in 20 or 40 ml foam-plugged glass vials and starved for 24 hours. A 5 mm diameter lettuce disk with either 104, 105 or 106 spores of L. dichroplusae each in aqueous suspension was then administer to each grasshopper. Only those that consumed the entire disk were considered inoculated, and were kept for 30 days, checking regularly for infection development through dissection and homogenization. Controls were identical but without adding spores to the lettuce disks. Table 1 shows the bioassays performed.
Table 1. Per os experimental inoculations (bioassays) with spores of Liebermannia dichroplusae on its natural host Dichroplus elongatus.
Host nymphal Number of inocu- Dose per Infection de-
instar or stage lated individuals individual velopment
I 24 104 No
I 23 105 No
I, II 20 106 No
II 30 105 No
II 30 106 No
II, III 24 105 No
III 20 105 No
III 32 106 No
IV 15 105 No
IV, V 25 106 No
V 28 106 No
Adult 20 106 No
Results
Only six individuals of D. elongatus had infections. Three of them were infected with P. locustaealone, two were infected with L. dichroplusae alone, and one individual had a co-infection with both microsporidia (Fig. 1). Therefore both pathogens occurred at low prevalence, P. locustae at 4.6 % and L. dichroplusae at 3.4 %. Spore loads per individual were 1.7*109, 8.6*108 and trace as defined by Henry (1972) for P. locustae, 4.1* 108 and 3.4* 107 for L. dichroplusae, and 1.0* 106 and 2.9* 105 for P. locustae and L. dichroplusae, respectively in the mixed infection. Heaviest infection by each pathogen alone (1.7* 109 for P. locustae, 4.1 * 108 for L. dichroplusae) showed typical internal signs of infection following ventral dissection. Creamy color with some pink tinge and heavily hypertrophied fat body in the case of P. locustae (Fig. 2), and whitish, also greatly hypertrophied Malpighian tubules in the case of L. dichroplusae (Fig. 3). No obvious internal signs of infection by either of the pathogens were observed in the mixed infection. All bioassays gave negative results (i.e. no infection development).
Discussion
This is the first detection of the microsporidia P. locustae and L. dichroplusae sharing the same individual host (i.e. co-infection). Since both pathogens are found with relative frequency in D. elon-gatus of the Pampas and considering that it took
Fig. 1. Mature spores of Paranosema locustae (larger ones) and Liebermannia dichroplusae (smaller ones) from the co-infection in Dichroplus elongatus. Scale bar: 5 pm.
approximately three decades of monitoring of thousands of D. elongatus from nearly 100 localities (Lange, 2003; Bardi et al., 2012) prior to finding the co-infection here reported it might be assumed that such an event is highly unusual. Indeed, natural mixed infections by entomopathogenic microspo-ridia species appear to be a rare phenomenon possibly due to competition issues (Solter, 2014). A possible antagonistic interaction was previously hypothesized between P. locustae and L. dichroplusae (Lange and Cigliano, 2005), as it was also suggested for P. locustae and an undescribed micro-sporidium in North America (Streett and Henry 1984), because by 2003 at only one locality (Carhue, western Buenos Aires province) and for just two seasons both pathogens were found to coexist in D. elongatus, albeit in different individuals (Lange, 2003). Since then, further monitoring revealed the coexistence of P. locustae and L. dichroplusae in populations of D. elongatus in the surroundings of five additional localities in the Pampas, also in western Buenos Aires province (Alamos, Blancagrande, Casbas, Espartillar, Trenque Lauquen) but without the occurrence of mixed infections. The co-infection herewith reported shows that there is no absolute antagonism between P. locustae and L. dichro-plusae but possibly a more subtle interaction that under a very narrow set of opportunistic-based type of conditions may allow simultaneous occurrence in a single host. Unfortunately, at the moment we cannot test in the laboratory the interactions between both microsporidia simul-taneously within the host due to the inability of inducing
Fig. 2. Ventrally dissected adult of Dichroplus elongatus depicting heavily infected fat body tissue with Paranosema locustae. Scale bar: 20 mm.
infections with L. dichroplusae. We speculate that given the characteristics inherent to both pathogens a conceivable scenario would be that the individual of D. elongatus was already infected with L. dichroplusae when it contracted infection by P. locustae. Horizontal transmission is highly efficient, spore loads are frequently heavy, and host range is unusually wide in P. locustae, whereas vertical transmission seems to predominate, spore loads are seldom too heavy, and host range is monospecific in L. dichroplusae. In any case, we feel that the rarity of the finding merits publication.
Acknowledgments
To Dr. S. Plischuk for discussing an earlier version of the manuscript.
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Address for correspondence: Carlos E. Lange. CEPAVE, Boulevard 120 s/n entre Av. 60 y Calle 64, La Plata (1900), Argentina; e-mail: carlosl@cepave.edu.ar.
