Naturalized, newly-associated microsporidium continues causing epizootics and expanding its host range Текст научной статьи по специальности «Биологические науки»

Protistology 14 (1), 32-37 (2020)

Protistology

Naturalized, newly-associated microsporidium continues causing epizootics and expanding its host range

Carlos E. Lange12, Yanina Mariottini3, Santiago Plischuk1, and María Marta Cigliano14

1 Centro de Estudios Parasitológicos y de Vectores (CEPAVE), CCT La Plata CONICET - Universidad Nacional de La Plata (UNLP), Argentina

2 Comisión de Investigaciones Científicas de la Provincia de Buenos Aires (CICPBA), Argentina

3 Instituto Multidisciplinario sobre Ecosistemas y Desarrollo Sustentable (UNICEN-CICPBA), Tandil, Argentina

4 Museo de La Plata, División Entomología, Facultad de Ciencias Naturales, Universidad Nacional de La Plata (UNLP), Argentina

| Submitted January 4, 2020 | Accepted January 24, 2020 |

Summary

Examination of grasshoppers sampled at two sites, one in western Pampas and one in northwestern Patagonia, both in Argentina, revealed the occurrence of two new epizootic events of the allochthonous microsporidium Paranosema locustae which became naturalized in grasshopper communities of those areas after introductions from its native land in North America in the late seventies-early eighties and mid-nineties, respectively. In the Pampas site, P. locustae was found infecting six out of eight grasshopper species collected at an overall prevalence of 38.8 % and a maximum of 50 %. In the Patagonia site, P. locustae was detected in four out of six species at an overall prevalence of 10.5 % and a maximum of 34.3 %. Two of the species affected, one in the Pampas, the gomphocerine Amblytropidia australis, and one in Patagonia, the melanopline Dichroplus vittigerum, constitute new host records for P. locustae, expanding its field host range in Argentina to 24 species of grasshoppers. In addition, bioassays were performed on three other grasshopper species selected based on several criteria. The tristirid Bufonacris claraziana and the gomphocerine Borellia bruneri turned out to be not susceptible to P. locustae while the melanopline Scotussa cliens was highly susceptible. All in all, results highlight not only the uniqueness of P. locustae among the Microsporidia that allowed for its development as a biocontrol agent but also the suitability of grasshopper communities in the Pampas and Patagonia to fully express such uniqueness, possibly even more so as an allochthonous parasite against autochthonous novel hosts.

Key words: Argentina, biocontrol agent, grasshoppers, Paranosema locustae, prevalence

doi:10.21685/1680-0826-2020-14-1-4 © 2020 The Author(s)

Protistology © 2020 Protozoological Society Affiliated with RAS

Introduction

An allochthonous species is considered to be naturalized if after a prolonged period of time and a number of generations it is still established or expanding in the new area, not requiring further inputs for survival and reproduction. Ifthe species is parasitic and the hosts affected are autochthonous, then new parasite-host associations occur (Perlman and Jaenike, 2003). The more species ofhosts that are susceptible to the novel parasite in a community the greater the chances are of naturalization (Bonsall, 2004). The fate of naturalized parasites on naive, newly-associated hosts tends to be unpredictable but the lack of co-evolutionary experience of the new associations may result in enhanced impact or unexpected outcomes.

The microsporidium Paranosema locustae has been developed as a long-term grasshopper biocon-trol agent (Henry, 2017; Lange and Sokolova, 2017) and it is known to occur in grasshopper communities of areas in northwestern Patagonia (Neuquen and Chubut provinces) and Pampas regions (Buenos Aires and La Pampa provinces) of Argentina since it was re-isolated in 1991 (Lange, 1992) after its introduction from North America more than forty years ago in the Pampas and Chubut (Lange and Cigliano, 2005; Bardi et al., 2012) and 24 years ago in Neuquen (Lange and Azzaro, 2008; Lange, 2010). Paranosema locustae, an intracellular microparasite, develops in the grasshoppers adipocytes of the fat body, depriving the host of vital energy for development and reproduction and causing not an acute disease but rather a chronic, debilitative illness characterized by numerous sub-lethal effects. These include reductions of fecundity and longevity, delays in development and molting abnormalities, decrease of activity and lethargy, reduction of food consumption, flying inability or difficulties, disruption of aggregation behavior, and alteration of phase transformation (Lange and Cigliano, 2005; Fu et al., 2010; Feng et al., 2015).

In the present contribution we report the detection of two new epizootic events of P. locustae, the finding of field infections in two grasshopper species not previously recorded to be susceptible, and the result of laboratory bioassays against three grasshopper species of unknown susceptibility

Material and methods

Field grasshopper samples consisted of all individuals captured alive with three hundred sweeps of

entomological nets at each of two grassland sites, one in the Pampas region on February 11, 2017 (15 km North of Santa Rosa, La Pampa province; 36°28'15.85"S, 64°16'47.03"W) and the other in north-western Patagonia on February 3, 2016 (3 km SW ofLoncopue, Neuquen province; 38°05'30.50"S, 70°39'28.82"W), areas where P. locustae is known to be established in grasshopper communities since long ago (Lange and Azzaro, 2008; Bardi et al., 2012). In the Pampas site, a total of286 grasshoppers belonging to eight species (Amblytropidia australis, Baeacris pseudopunctulata, Borellia bruneri, Dich-roplus elongatus, D. pratensis, D. vittatus, Rhamma-tocerus pictus, Staurorhectus longicornis) were collected. In the Patagonia site, 496 grasshoppers of six species (Borellia bruneri, Dichroplus elongatus, D. maculipennis, D. vittigerum, Scotussa lemniscata, Scyl-linula signatipennis) were obtained. Soon after collection, the samples were frozen (-32 °C) until processing. Individual examination of each grasshopper was done either by organ/tissue scrutiny after ventral, longitudinal dissection and/or whole-body homogenization in double distilled water followed by phase contrast microscopy (400x, 1000 x) as already described in Lange and Cigliano (2019). Changes in the gross appearance of the grasshopper's fat body infected with P. locustae are a pathognomonic sign (Henry and Oma, 1981; Lange and Cigliano, 2019) and both the size and shape of spores and type of developmental stages of P. locustae are easily distinguished under phase contrast microscopy from all known autochthonous grasshopper microsporidia, all ofthem belonging to genus Liebermannia (Lange, 2003, 2010; Sokolova et al., 2009; Lange and Cigliano, 2019).

Bioassays in the form of oral experimental inoculations as described by Mariottini and Lange (2014) and Lange and Cigliano (2019) were performed on juvenile individuals (third and fourth nym-phal instars) of three species of grasshoppers not previously challenged with P. locustae. Source spores employed in the bioassays as aqueous suspensions at a dose of 105 per each challenged insect were obtained from infections in the establishment area of the Pampas (Bardi et al., 2012). Grasshopper species challenged were B. bruneri, Scotussa cliens, and Bufonacris claraziana. Individuals inoculated, 40 B. bruneri, 43 S. cliens, and 58 B. claraziana, were F1 laboratory generation of grasshoppers collected as adults in the vicinity of Laprida county, southern Pampas (Buenos Aires province), La Plata and Lobos counties, northeastern Pampas (Buenos Aires province), and Cushamen, northwestern Patagonia

Table 1. Host range and prevalence of the microsporidium Paranosema locustae in grasshoppers collected in the Pampas site (15 km North of Santa Rosa, La Pampa province).

Subfamily of Acrididae Species Infected/ Collected Prevalence (%)

Melanoplinae Baeacris pseudo-punctulatus 2/4 50.0

Dichroplus elongatus 2/6 33.3

Dichroplus pratensis 85/203 41.9

Dichroplus vittatus 1/5 20.0

Gomphocerinae Amblytropidia australis 1/10 10.0

Borellia bruneri 0/4 00.0

Rhammatocerus pictus 0/1 00.0

Staurorhectus longicornis 10/53 18.9

(Chubut province), respectively. Criteria for selecting these three species for performing bioassays were as follows. Borellia bruneri belongs to a subfamily of Acrididae, the Gomphocerinae, whose specific members tend to be susceptible to P. locustae (Lange, 2005). However, in spite of B. bruneri being relatively common in some areas where P. locustae became naturalized, it has never been found infected. In outbreak years, B. bruneri is considered an actual agricultural pest in the Pampas of Uruguay and has the potential to become so in areas of the Argentine Pampas and Patagonia (Cigliano et al., 2014). Scotussa cliens also belongs to a subfamily, the Melanoplinae, whose members are possibly the most susceptible to P. locustae. Since S. cliens overwinters as nymphs (Liebermann, 1952) it might have the ability to carry the disease from one season to the next. The Tristiridae B. claraziana, a heavy-built, large (up to 36.7 mm long) apterous grasshopper endemic by Patagonia causes serious rangeland forage losses when outbreaks occur (Cigliano et al. 2014). None to the 25 known species of tristirids inhabiting South America (Cigliano et al., 2019) have ever been challenged with P. locustae which means that there is no clue about the intrinsic suitability of tristirids for infection development.

Results

In the Pampas site, P. locustae was found infecting six out of the eight species collected in an overall prevalence of 38.8 % (Table 1). The highest per species prevalence was recorded in B. pseudo-

Table 2. Host range and prevalence of the microsporidium Paranosema locustae in grasshoppers collected in the Patagonia site (3 km SW of Loncopué, Neuquén province).

Subfamily of Acrididae Species Infected/ Collected Prevalence (%)

Melanoplinae Dichroplus elongatus 22/83 26.5

Dichroplus maculipennis 23/67 34.3

Dichroplus vittigerum 2/45 4.4

Scotussa lemniscata 5/56 8.9

Gomphocerinae Borellia bruneri 0/160 00.0

Scyllinula signati-pennis 0/85 00.0

punctulata (50 %) and the lowest in al. australis (10 %). However, prevalence of 41.9 % in D. pratensis and 18.9 % in S. longicornis are the ones sustained on considerable sample size, 203 and 53 individuals, respectively.

In the Patagonia site, P. locustae was found infecting four out ofthe six species collected in an overall prevalence of 10.5 % (Table 2). The highest per species prevalence was 34.3 % in D. maculipennis (n=67) and the lowest was 4.4 % in D. vittigerum (n=45).

Infections in both A. australis and D. vittigerum constituted the first records of P. locustae parasitizing such host species. None of the experimentally inoculated B. bruneri and B. claraziana developed infections while infection development occurred in 97.7 % of inoculated S. cliens.

Discussion

As pointed out by Lange and Sokolova (2017), P. locustae exhibits a combination of traits that makes it a remarkable, unique microsporidium: (i) it has an unusual wide host range among acridomorphs (up until this contribution 124 species were known to be susceptible worldwide), (ii) per os horizontal transmission is highly efficient and it is complemented with a vertical route through the female line, (iii) infections tend to be chronic rather than acute, typically allowing for the production of heavy spore loads per infected grasshopper host, and (iv) spores maintain viability at freezing temperatures for extended periods of time (years). These attributes confer P. locustae an exceptional ability for widespread occurrence and long-term persistence in grasshopper communities having susceptible spe-

Table 3. Host range of the microsporidium Paranosema locustae in grasshoppers of Argentina.

Family Subfamily Species Field infection Exprimental infection Challenged but no infection

Baeacris pseudopunctulata X

Baeacris punctulata X X

Dichroplus conspersus X

Dichroplus elongatus X X

Dichroplus maculipennis X X

Dichroplus pratensis X X

Dichroplus schulzi X

Melanoplinae Dichroplus vittatus X

Dichroplus vittigerum X

Leiotettix pulcher X

Neopedies brunneri X

Ronderosia bergii X X

Ronderosia forcipata X

Acrididae Scotussa cliens X

Scotussa daguerrei X

Scotussa lemniscata X

Amblytropidia australis X

Borellia bruneri X

Euplectrotettix schulzi X

Gomphocerinae Laplatacris dispar X

Rhammatocerus pictus X

Sinipta dalmani X

Staurorhectus longicornis X

Cyrtacanthacridinae Schistocerca cancellata X

Oedipodinae Trimerotropis pallidipennis X X

Copiocerinae Aleuas lineatus X X

Acridinae Allotruxalis gracilis X

Diponthus argentinus X

Romaleidae Romaleinae Staleochlora viridicata X

Tropidacris collaris X

Zoniopoda tarsata X

Tristiridae Tristirinae Bufonacris claraziana X

cies. In spite of this, occurrence appears not as wide as might be expected in its native land in North America (Arizona, Colorado, Idaho, Minnesota, Montana, North Dakota, Oregon, and Wyoming in the USA as well as southwestern Canada; Lockwood and Larsen, 1988) and prevalence is known to be normally less than 1% with rare peaks of 5% (Henry and Oma, 1981; Ewen, 1983). Despite heavy survey efforts in North America only once a natural epizootic (i.e., not following a field application) was detected (Henry, 1972). It might be possible that such low rates of natural prevalence where P. locustae is autochthonous would be somehow related with the longtime association between P. locustae and North American grasshoppers, where

pathogen and hosts probably co-evolved towards a sort of "win-win" equilibrium for both parties. The high prevalence and wide host range here reported in samples at both the Patagonia and Pampas sites not only corroborate the occurrence of significant epizootics in grasshopper communities in areas of naturalization of P. locustae in Argentina but also suggest that such an outcome might be related with the novel pathogen-hosts associations generated. We think it is relevant to report about these two new epizootic events for several reasons: i) as mentioned above, the apparent extreme rarity of such events in North America, ii) the fact that similar events were observed long ago in other four different localities in the Pampas (Lange and de Wysiecki,

2005) but at a time when monitoring surveys were much more intense. The intensity of surveys was relatively high until 2011 (Bardi et al., 2012) but was severely relaxed since then which might indicate that epizootics could be a more common outcome in more recent years, ii) although a quantitative assessment of the effect of naturalized P. locustae on grasshopper communities is not possible due to a lack of pre-introduction grasshopper abundance data, epizootics might explain into some extent why the frequency and severity of pest grasshopper outbreaks appear to have diminished in areas of naturalization.

The detection of infections in A. australis and D. vittigerum expands the known field host range of P. locustae in Argentina to 24 grasshopper species (Table 3). In a sense, these findings should not be considered as unexpected because both A. australis and D. vittigerum belong to subfamilies of Acrididae, the Gomphocerinae and Melanoplinae, respectively, which are of known susceptibility to P. locustae (Lange, 2005). However, the detection of field infections are the actual or ultimate confirmation of susceptibility as opposed to infections induced in the laboratory where unknown ecological barriers or other factors may be bypassed by the forced inoculation procedure.

Results from the bioassays felt within our presumptions. As should be expected from a Melano-plinae, Scotussa cliens resulted to be highly susceptible which means that in a field situation scenario infected overwintering nymphs could be able to carry the pathogen from one season to the next and conceivably be the source of a new appearance ofthe disease in a given area and eventually an epizootic. S. cliens is widespread and common in the Pampas (Cigliano et al., 2019). The negative outcome of the bioassays (i.e. lack of infection development) in B. bruneri concurs with the fact that this species has never been found infected in the field in spite of belonging to a subfamily (Gomphocerinae) of recognized general susceptibility. Although close phylogenetic relatedness seems to be generally a predictor of susceptibility to P. locustae among the Acrididae exceptions clearly do occur and ultimate susceptibility seems to be at the host species level. Absence of infections in the bioassays on B. claraziana is not surprising considering the phylogenetic distance between Tristiridae and Acrididae (Song et al. 2015).

Our results highlight not only the uniqueness of P. locustae among the Microsporidia that allowed

its development as a biocontrol agent but also the suitability of grasshopper communities in the Pampas and Patagonia to fully express the combination of traits that account for such uniqueness, possibly even more so as an allochthonous parasite against novel hosts rather than as an autochthonous one in co-evolved hosts. Lange and Cigliano (2005) and Bardi et al. (2012) discussed at length the potential of P. locustae to alter the structure and dynamics of nanve grasshopper communities in Argentina through differences on the inherent susceptibility of the specific components, even singling out species that might be at risk due to P. locustae as a threat, and our new findings here reported reinforce that particular quality of this remarkable microsporidium.

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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