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Ecological vs physiological host specicity:
the case of the microsporidium
Nosema pyrausta (Paillot) Weiser, 1961
Yuri S. Tokarev1, Darya S. Kireeva1, Anastasia N. Ignatieva1,
Aleksander A. Ageev2, Aleksei V. Gerus1, Olga N. Yaroslavtseva3,
Anastasia G. Kononchuk1, Julia M. Malysh1
1 All-Russian Institute of Plant Protection, 3 Podbelskogo highway, Pushkin, St. Petersburg, 196608,
Russia
2 Center of Forest Pyrology, All-Russia Research Institute of Silviculture and Mechanization of Forestry,
42 Krupskoy st., Krasnoyarsk, 660062, Russia
3 Institute of Systematics and Ecology of Animals SB RAS, 11 Frunze st., Novosibirsk, 630091, Russia
Corresponding author: Julia M. Malysh (ymalysh@vizr.spb.ru)
Academic editor: R. Yakovlev| Received 25 June 2022| Accepted 6 July 2022 |Published 9 September 2022
http://zoobank.org/EB9C3099-F39A-4479-BD32-3033DF19287F
Citation: Tokarev YuS, Kireeva DS, Ignatieva AN, Ageev AA, Gerus AV, Yaroslavtseva ON, Kononchuk AG,
Malysh JuM (2022) Ecological vs physiological host specicity: the case of the microsporidium Nosema pyrausta
(Paillot) Weiser, 1961. Acta Biologica Sibirica 8: 297–316. https://doi.org/10.14258/abs.v8.e19
Abstract
e microsporidium Nosema pyrausta (Paillot) Weiser, 1961 plays an important role in the mortality
of the European corn borer Ostrinia nubilalis (Hübner, 1796), and shows high virulence to the beet
webworm Loxostege sticticalis (Linnaeus, 1761). In contrast, the greater wax moth Galleria mellonella
(Linnaeus, 1758) and the gypsy moth Lymantria dispar (Linnaeus, 1758) are referred to as resistant
hosts, slightly susceptible to this microparasite. e goal of the present study was to test N. pyrausta
against a broad range of lepidopteran species with dierent taxonomy, physiology, and ecology. e
susceptibility to N. pyrausta spores uctuated greatly among members of various families and super-
families of Lepidoptera. As many as 13 species tested were found to be refractory (not able to support
the development of the microsporidium), including three species of Yponomeutoidea, four species of
Papilionoidea, one species of Pyraloidea, two species of Bombycoidea, and three species of Noctuoi-
dea. e species found to be susceptible (with a high proportion of specimens displaying developed
infection) included: Evergestis forcalis (Linnaeus, 1758) (Crambidae), Aglais urticae (Linnaeus, 1758)
(Nymphalidae), and Dendrolimus sibiricus Chetverikov, 1908 (Lasiocampidae). e species newly
found to be highly susceptible (high proportion of infected insects accompanied with high levels of
Acta Biologica Sibirica 8: 297–316 (2022)
doi: 10.14258/abs.v8.e19
http://journal.asu.ru
Copyright Yuri S. Tokarev et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
RESEARCH A RTICLE
298Yuri S. Tokarev et al. / Acta Biologica Sibirica 8: 297–316 (2022)
early mortality) were: Spodoptera exigua (Hübner, 1808) (Noctuidae) and Aglais io (Linnaeus, 1758).
Large quantities of spores can be produced in vivo using substitute laboratory host A. urticae. ese
results conrm previous observations that physiological host range of microsporidia (observed under
experimental conditions) is broader than the ecological one (observed in nature).
Keywords
Microsporidia, microbial control, bioassay, virulence, lepidopteran pest
Introduction
Microsporidia are obligate intracellular eukaryotic parasites belonging to a certain
phylogenetic lineage, closely related to Fungi (Bass et al. 2018; Corsaro et al. 2019).
ey are ubiquitous as pathogens of animals and particular species are virulent to
their insect hosts (Issi 2020) and therefore are important for natural density regu-
lation and pest control (Franz and Huger 1970; Lipa and Madziara-Borusiewicz
1976; McManus and Solter 2003; van Frankenhuyzen et al. 2007; Hopper et al. 2016;
Frolov 2019; Andreeva et al. 2021; Malysh et al. 2021). One recent example of suc-
cessful application of microsporidia under eld conditions is that of Myrmecomorba
nylanderiae Plowes, Becnel, LeBrun, Oi, Valles, Jones, Gilbert, 2015 suppressing lo-
cal populations of the tawny crazy ant Paratrechina (Nylanderia) fulva (Mayr, 1862)
in North America (LeBrun et al. 2022). In Lepidoptera, several genera of micro-
sporidian parasites are found (Canning et al. 1985; Cali and Garhy 1991; Andreadis
et al. 1996; McManus and Solter 2003; Malysh et al. 2013, 2018). Among those, one
of the most abundant groups is the genus of Nosema with the type species Nosema
bombycis Nägeli, 1857 from the silkworm Bombyx mori Linnaeus, 1758 (Bomby-
coidea: Bombycidae), which suers devastating epizootics of this pathogen under
conditions of laboratory and industrial mass rearing (Bhat et al. 2009). One of the
closest relatives of this microsporidium is Nosema pyrausta (Paillot) Weiser, 1961, a
widespread pathogen of the European corn borer Ostrinia nubilalis (Hübner, 1796)
(Lepidoptera: Crambidae). Numerous observations indicate that in North Amer-
ica, microsporidia infections play an important role in regulation of O. nubilalis
population density, causing regular epizootics under eld conditions (Lewis et al.
2009; Zimmermann et al. 2016). Meanwhile, epizootics in European populations
of Ostrinia moths are less frequent and the average prevalence level typically does
not exceed 10 % (Pelissie et al. 2010; Malysh et al. 2011; Grushevaya et al. 2018).
is discrepancy can be explained by genetic dierences of the European and North
American isolates of the parasite (Tokarev et al. 2015) as well as other ecological
factors diering between the primary (Europe) and the secondary (North America)
areas of the pest.
Host range of a given microsporidium species is an important indicator of its
ability to circulate in nature and inuence insects other than the type host, as well as
its potential for practical implications (Solter et al. 1997, 2010). e beet webworm
Ecological vs physiological host specicity: the case of the microsporidium Nosema pyrausta (Paillot) Weiser, 1961299
Loxostege sticticalis (Linnaeus, 1761), a notorious polyphagous insect belonging to
the same family Crambidae as Ostrinia, was found to be highly vulnerable to N.
pyrausta and demonstrates high mortality rates even at low dosages of the patho-
gen (Malysh et al. 2021). On the contrary, the greater wax moth Galleria mellon-
ella (Linnaeus, 1758) (Pyraloidea: Pyralidae) and the gypsy moth Lymantria dispar
(Linnaeus, 1758) (Noctuoidea: Erebidae) are considered to be the resistant hosts.
In these two insect species, the mean dosage of 2 million (mln) spores per second
instar larva caused infection prevalence at the level of 0-5 %, which could be fur-
ther augmented only by a combination of additional immunosuppressive factors
(Tokarev et al. 2018; Kononchuk et al. 2021). Information concerning susceptibil-
ity of other lepidopteran species to this pathogen could not be found, except for
one study where few larvae of the common buckeye Junonia coenia Hübner, 1822
were tested, resulting in 50 % infection prevalence level (Hall 1952). Meanwhile,
understanding of host ranges and the factors governing host specicity of insect
pathogens, including microsporidia, is inevitable for exploration of patterns of their
natural distribution and for prediction of interplay with other ecosystem compo-
nents upon introduction into new habitats (Jeords et al. 1989; Onstad 1993; Solter
et al. 1997, 2010; Vilcinskas 2019; Issi 2020). Before such species will be considered
for application as microbial control agents, their virulence against crop and forest
pests, as well as interactions with non-target entomofauna, should be carefully ex-
amined to achieve desirable levels of pest management ecacy and to ensure safety
for natural biodiversity.
To explain why dierent insect species vary in their susceptibility levels to a cer-
tain microsporidium, several reasons can be assumed. First, host specicity of the
microparasite may play a certain role, when closely related insects are susceptible to
a certain microsporidium while distant host taxa are less prone to this microparasite
species. In fact, L. sticticalis belonging to the same family Crambidae and subfamily
Pyraustinae as Ostrinia shows high susceptibility to the microsporidium from the
latter host, as opposed by resistant Galleria and Lymantria from other high-level
taxa (see above). Second, insect body size may be of importance, as younger (and
smaller) instars are usually more susceptible to microsporidia and other microbes
as compared to the older (and bigger) ones (Vogelweith et al. 2013). is negative
correlation between the body size and infection susceptibility observed within the
course of individual development of a certain species may also have impact when
dierent insect species are compared. In particular, Loxostege is smaller than Os-
trinia which in turn is smaller than Galleria and Lymantria; and susceptibility to
N. pyrausta is gradually decreasing in this row. ird, feeding behaviour should de-
ne chemical composition of the insect gut juice so that the microsporidium spore
activation is aected (Issi et al. 2005). A polyphagous host is expected to have a
non-specic set of stimuli which potentially might activate the microsporidia spore
extrusion in the midgut lumen. Hence, the polyphagous herbivores Ostrinia and
Loxostege seem to be more suitable as a host for a certain species of microsporidia
300Yuri S. Tokarev et al. / Acta Biologica Sibirica 8: 297–316 (2022)
as compared to the dendrophilic phyllophagous larvae of Lymantria and wax-con-
suming Galleria.
e goal of the present study is to test these assumptions experimentally using
administration of N. pyrausta spores against a broad range of lepidopteran insect
species with dierent taxonomy, physiology, and ecology.
Material and methods
Propagation of the microsporidium
Nosema pyrausta spores were propagated in O. nubilalis under laboratory condi-
tions as described earlier (Grushevaya et al. 2018). Batches of spores were isolated
from the host pupae, washed with distilled water by centrifugation at 1000 g for 5
min and stored prior to experimental assays as pellets in water refrigerated for 1-3
months, which should not impair signicantly their infective potential (Malysh et
al. 2021).
Insects
For experimental infection, second instars of lepidopteran insects were used either
collected in nature and assayed directly, propagated as a temporary laboratory cul-
ture, or taken from a permanent laboratory culture.
Directly assayed insects collected in nature as the second instar larvae on their
type host plants were the ermine moths Yponomeuta evonymella (Linnaeus, 1758)
and Yponomeuta malinellus Zeller, 1838, the small tortoiseshell Aglais urticae (Lin-
naeus, 1758) and the European peacock Aglais io (Linnaeus, 1758) in St. Petersburg,
the black-veined white Aporia crataegi (Linnaeus, 1758) in Novosibirsk and the fall
webworm Hyphantria cunea (Drury, 1773) in Krasnodar Area. e cabbage white
Pieris brassicae (Linnaeus, 1758) was available at all developmental stages during
the vegetation season in St. Petersburg, but its larval population is constantly infest-
ed by the parasitoid, Cotesia glomerata (Linnaeus, 1758), at high prevalence levels.
For this reason, eggs were collected in nature and hatched larvae were reared until
second instar larvae under laboratory conditions. ese insects were fed with fresh
leaves of their type host plants or other available species: bird cherry for Y. evony-
mella, apple for Y. malinellus, nettle for Aglais spp., plum for A. crataegi, ash-leaved
maple for H. cunea, and cabbage for P. brassicae.
Temporary cultures were also established to obtain the second instar larvae of
the lial generation reared under laboratory conditions. ese species included
insects either caught as adults (the small white Pieris rapae (Linnaeus, 1758), the
green-veined white Pieris napi (Linnaeus, 1758), and the Indian our moth Plodia
interpunctella (Hübner, 1813) in St. Petersburg) or sampled as the last instar larvae
Ecological vs physiological host specicity: the case of the microsporidium Nosema pyrausta (Paillot) Weiser, 1961301
(the diamondback moth Plutella xylostella (Linnaeus, 1758) in St. Petersburg, the
cotton bollworm Helicoverpa armigera (Hübner, 1808) in Krasnodar Area, the cab-
bage moth Mamestra brassicae (Linnaeus, 1758) in Novosibirsk Region and the Si-
berian moth Dendrolimus sibiricus Chetverikov, 1908 in Krasnoyarsk Region). e
larvae were fed either with their type host plants, such as cabbage leaves (P. rapae, P.
napi, P. xylostella, M. brassicae), and r branches (D. sibiricus), or meridic diet (H.
armigera, P. interpunctella).
As many as three insect species were available as the permanent laboratory cul-
tures. e silkworm B. mori rst instar larvae were purchased from Research Sta-
tion of Sericulture (Stavropol Area) and maintained at the facilities of Slavyansk
Experimental Station of Plant Protection (Krasnodar Area) on mulberry leaves. e
tobacco hornworm Manduca sexta Linnaeus, 1763 eggs were purchased from the
group of companies “T-RexFoods” (Moscow) and reared on a commercial meridic
diet (“T-RexFoods”). e beet armyworm Spodoptera exigua (Hübner, 1808) was
propagated at the facilities of the University of Silesia on a meridic diet.
Experimental infection
e second instars were used for all experimental treatments. e groups of 21-30
larvae of each insect species were maintained for 4-24 hours without feed. en
the starved insects were provided with N. pyrausta-contaminated feed. e plant
leaves (or the r needles) were evenly covered with the spore suspension on both
sides and le for air-drying for several minutes. When necessary, the leaf cuticle was
scratched by a pin to facilitate moistening of the leaf surface. For the insects main-
tained on the meridic diets, a diet portion was mixed with the spore suspension. In
all cases, the amount of spores was adjusted to provide the mean dosage of 1 mln
of spores per larva, taken that 90-100 % of the contaminated feed portion is con-
sumed. Aer contaminated feed consumption, the groups of larvae were split into
three equal groups to represent the repetitions, or maintained as a single repetition.
e pathogen-free feed was provided to the insects for the rest of the experiment.
Control insects were treated similarly but without addition of the microsporidium
spores. Mortality was screened on a daily basis, cadavers were dissected, and inner
tissues examined using light microscopy. In A. urticae, an additional experiment
was performed using the dosage lower by an order of magnitude (0.1 mln spores/
larva). In A. crataegi, two dosages were also used, 0.1 mln and 1 mln spores/larva,
respectively. In this experiment, ten larvae in each variant, including control, were
dissected at 30th day post treatment (d.p.t.). e rest of the insects were transferred
to +4°C for hibernation for four months and then dissected. For M. sexta, P. brassi-
cae and P. rapae, additional series of experiments were performed using insects fed
with the diet (M. sexta) with addition of 1 % phenylthiourea (PTU) or the cabbage
leaves (P. brassicae, P. rapae) sprayed with 1 % PTU one day prior to experimental
infection with microsporidia. is was done to increase insect susceptibility to the
non-specic microsporidium infection (Tokarev et al. 2018). Aer one day feeding
302Yuri S. Tokarev et al. / Acta Biologica Sibirica 8: 297–316 (2022)
on the PTU-treated fodder, the insects were treated with the parasite’s spores as
above. To calculate the spore loads in infected insects, the homogenates were pre-
pared from the individual specimens and amount of spores was quantied using a
haemocytometer.
Molecular genetic diagnosis
Samples of infected tissues or isolated spores were subjected for DNA extraction,
PCR with microsporidia-specic primers 18f:1047r and sequencing to conrm the
species diagnosis of the parasite (Weiss and Vossbrinck 1999; Malysh et al. 2019).
When necessary, the primers LepF1:LepR1 specic for mitochondrial cytochrome
oxidase subunit I (COI) were used for barcoding of Lepidoptera (Hebert et al.
2004). Sequences were analyzed and compared using BioEdit soware (Hall 1999)
and BLAST utility at the NCBI server (Altschul et al. 1990).
Statistical analysis
Survival analysis, including estimation of median survival time (LT₅₀), was estimat-
ed using Kaplan-Meier procedure followed by log rank test in SigmaPlot 12.5 (Systat
Soware Inc., San Jose, CA, 2011).
Results
Among the three species assayed in Yponomeutoidea, neither Yponomeutidae (the
two species of the ermine moths) nor Plutellidae (the diamondback moth) became
infected with N. pyrausta aer feeding with the spores at early larval stage and
maintainance until pupation or adult emergence.
Among Papilionoidea, four species of Pieridae and two species of Nymphalidae
were tested. None of the P. rapae, P. brassicae, and P. napi, including those pretreated
with PTU for immunosuppression (P. rapae and P. brassicae) became infected. How-
ever, in the group of P. rapae, one larva out of 30 turned out to possess morphol-
ogy (Fig. 1) drastically distinct from that of other Pieridae as the insects grew up.
is specimen did not pupate for as long as three months aer successful pupation
of all other insects in both experimental and control groups. Obviously, another
species was assayed accidentally alongside with P. rapae larvae. e adipose tissue
of this larva was loaded with Nosema-like spores. e tissue sample was used for
both parasite and host identication using molecular genetic tools. As a result, the
parasite was conrmed as N. pyrausta basing upon 100 % identity of the SSU rRNA
gene sequence (852 bp) to Genbank accession # HM566196. Meanwhile, the host
showed 100 % identity of 640 bp long COI sequence to the respective nucleotide
sequence (# GU828662) of the garden pebble moth, Evergestis forcalis (Linnaeus,
1758) (Lepidoptera: Crambidae).
Ecological vs physiological host specicity: the case of the microsporidium Nosema pyrausta (Paillot) Weiser, 1961303
In A. crataegi larvae, specimens positive for Nosema-like spores were found in
all treatment groups dissected both before and aer the hibernation, as well as in the
control, presumably indicating either successful infection with N. pyrausta, includ-
ing contamination of the control group, or natural infection with a microsporidium.
ese spores were elongated oval with blunt ends (Fig. 2A). eir length was in the
range of 3.3-4.5 (mean 3.8) µm, the width was 1.7-2.2 (mean 1.9) µm (number of
measured spores N=20). Meanwhile, the spores of the N. pyrausta isolate used for
the bioassays in the present study were oval with more tapered ends (Fig. 2B). e
N. pyrausta spore length was 3.0-3.9 (mean 3.4) µm, the width was 1.4-1.8 (mean
1.7) µm (N=20 spores). e prevalence level ranged between 20 and 40 % with an
average of 27.7±3.3 % (mean±SE, number of dissected insects N=67). In order to
identify the parasite, SSU rRNA gene fragment was sequenced, which showed 100
% identity (617 bp) to the respective sequence of Nosema sp. CmM2 (# KC836092)
from Cnaphalocrocis medinalis Guenée, 1854 (Lepidoptera: Crambidae). Other
most similar entries found in Genbank with 99.7 % sequence identity belonged to
N. furnacalis (# U26532), N. granulosis (# FN434087), and two isolates of uniden-
tied Nosema species from Spodoptera litura (Fabricius, 1775) (# LC422335) and
Operophtera bruceata Hulst, 1886 (# MG456600). On the other hand, identity to N.
pyrausta (# HM566196) and N. bombycis (# D85503) was below 98 %.
In A. urticae, mortality in control did not exceed 13 % during 25 days of the ex-
periment, and similar dynamics was observed in insects treated with 0.1 mln spores/
larva. On the contrary, the larvae treated with 1 mln spores/larva displayed mortal-
ity of 30 % and 48 % at 10th and 25 d.p.t., respectively. ese values were signicantly
dierent from both the lower dosage and the control groups (Fig. 3). e survived
larvae both in control and treatment groups successfully pupated on 15-20th d.p.t.
Notably, 100 % of perished larvae and survived pupae dissected at 10-30th d.p.i.
showed presence of spores in treatment groups, as opposed to the control group.
Multiple infection loci lled with prespore stages and mature spores were found in
salivary glands and adipose tissue (Fig. 4). e sequencing of SSU rRNA of selected
specimens conrmed the diagnosis of N. pyrausta infection as above. e pupae
from the experimental group infected with 1 mln spores/larva contained from 4 to
625 mln spores, with an average of 246±54.3 spores/pupa (N=12). When A. io was
Figure 1. Full-grown larva of Evergestis forcalis heavily infected with Nosema pyrausta.
Ruler division = 1 mm.
304Yuri S. Tokarev et al. / Acta Biologica Sibirica 8: 297–316 (2022)
assayed, this species also showed 100 % infection with N. pyrausta spore masses
(N=23), though exact mortality and spore load data were not collected. It can only
be noted, that on 7th d.p.t., as many as 12 out of 30 larvae perished (40.0±5.77 %)
with zero mortality in control. Among those perished larvae, microsporidia preva-
lence level reached 100 % and the spore load averaged 5.9±0.14 spores/larva.
Figure 2. Bright eld light microscopy of the spores of Nosema sp. from Aporia crataegi (A)
and Nosema pyrausta from Ostrinia nubilalis (B).
Figure 3. Mortality dynamics in Aglais urticae aer feeding with Nosema pyrausta at the
dosages of 0.1 or 1 million spores per second instar larva. Dierent letters indicate mortality
curves with signicantly dierent median lethal time at p<0.01. For raw data, see supple-
mentary material, Table 1.
Ecological vs physiological host specicity: the case of the microsporidium Nosema pyrausta (Paillot) Weiser, 1961305
In Pyraloidea, the only species assayed within the frames of the present study
was P. interpunctella, while data for other Pyralidae and Crambidae have been re-
trieved from published literature. In this stored grain pest, only a limited sample
of 13 larvae was available for N. pyrausta treatment assay, and 12 in control. Early
larval mortality within 10-14 d.p.t. reached as much as 25-33 % in both groups,
followed by pupation of the survivors within 15-45 d.p.t. In one pupa from the N.
pyrausta treatment group, formed by 34th d.p.t., few oval Nosema-like spores were
detected. In another pupa from 43rd d.p.t., both few Nosema-like spores and multi-
ple oval spores of smaller size in packets by 8 were observed, the latter referred to
as the octospores (Fig. 5). e Nosema-like spores measured 3.5 × 1.7 µm, length/
width ratio of 2.1 (N=18) while the octospores measured 2.5 × 1.5 µm, length/width
ratio of 1.7 (N=18). None of the control insects were infected (N=12). Sequencing of
SSU rRNA of both the Nosema-like spores and the octospores resulted in the iden-
tical reads, up to 802 bp long (#ON256647), with 99.4 % identity to the reference
sequence of Vairimorpha carpocapsae (Paillot, 1938) (# AF426104).
Figure 4. Bright eld light microscopy of Nosema pyrausta infection in salivary glands (A-
C) and adipose tissue (D) of the small tortoiseshell Aglais urticae with clearly seen prespore
developmental stages (pds), immature (is) and mature spores (ms).
306Yuri S. Tokarev et al. / Acta Biologica Sibirica 8: 297–316 (2022)
Table 1. Raw data on the mortality of the small tortoiseshell, Aglais urticae aer treatment
of II instar larvae with Nosema pyrausta spores at two dosages
Treatment,
spores/larva
Repetion # Sample
size
Number of insects perished on days 5-25
5 10 15 20 25
Control 1 10 0 1 1 0 0
2 10 0 0 0 0 0
3 10 0 0 2 0 0
0.1 mln 1 7 0 0 0 0 0
2 7 0 0 1 0 0
3 6 0 1 0 0 0
1 mln 1 10 0 3 0 1 2
2 10 0 4 0 0 0
3 9 0 2 0 1 1
Figure 5. Bright eld light microscopy of the Nosema-like spores (Nls) and the octospores
(os) of Vairimorpha cf carpocapsae detected in Plodia interpunctella.
In Lasiocampoidea, D. sibiricus was the only species assayed. In control, as
many as 6.7±4.55 % larvae perished by 3rd d.p.t., and mortality remained at this
level throughout the 60 days of the experiment. When challenged with N. pyrausta
spores (2 mln spores/larva), larval mortality steadily increased, reaching 33-71 %
within 10th-50th d.p.t. (Fig. 6). LT50 was 30.0±13.73 days. Both perished (starting
from 7th d.p.t.) and survived larvae were dissected, showing prevalence of the mi-
crosporidium at 92 % (N=26). Sequencing of amplicons obtained from ve selected
specimens (collected at 10th, 20th, 30th, 40th, and 50th d.p.t.) using microsporidia-
specic primers conrmed the diagnosis of N. pyrausta.
In Bombycoidea, two representatives of two respective families, B. mori (Bom-
bycidae) and M. sexta (Sphingidae) were assayed, both showing no changes in mor-
tality levels and pupation speed as compared to control, and no infection with mi-
crosporidia.
Ecological vs physiological host specicity: the case of the microsporidium Nosema pyrausta (Paillot) Weiser, 1961307
Figure 6. Mortality dynamics in Dendrolimus sibiricus aer feeding with Nosema pyrausta
at the dosage of 2 million spores per second instar larva. Dierent letters indicate mortality
curves with signicantly dierent median lethal time at p<0.01. For raw data, see supple-
mentary material, Table 2.
Table 2. Raw data on the mortality of the Siberian moth, Dendrolimus superans aer treat-
ment of II instar larvae with Nosema pyrausta at the dosage of 2 mln spores/larva
Treatment Repetition # Sample
size
Number of insects perished on days 5-60
5 10 15 20 25 30 35 40 45 50 55 60
Control 1 10 1 0 0 0 0 0 0 0 0 0 0 0
2 10 1 0 0 0 0 0 0 0 0 0 0 0
3 10 0 0 0 0 0 0 0 0 0 0 0 0
N. pyrausta 1 21 4 3 3 0 0 1 1 0 1 2 0 0
In Noctuoidea, representatives of two families were tested. Development of H.
cunea from Erebidae was not aected by N. pyrausta treatment challenging and
no infection was observed. Among Noctuidae, two species, H. armigera and M.
brassicae, were not aected by N. pyrausta challenging. On the contrary, S. exigua
displayed 100 % mortality of larvae challenged both with 1 and 0.1 mln spores/larva
within 10-12 d.p.t., which was by an order of magnitude higher as compared to con-
trol. Tissues of perished larvae were lled with N. pyrausta spores, as shown by light
microscopy and conrmed by PCR and sequencing as above.
308Yuri S. Tokarev et al. / Acta Biologica Sibirica 8: 297–316 (2022)
Discussion
Susceptibility to N. pyrausta varied greatly across families and higher rank taxa of
Lepidoptera. As many as 13 species assayed in the present study were found to be re-
fractory, i.e. not able to support development of the microsporidium, including the
representatives of Yponomeutoidea (3 species), Papilionoidea (4 species of Pieri-
dae), Bombycoidea (4 species), and Noctuoidea (2 species). Resistant hosts, which
could become infected only at low prevalence levels, were found in our previous
studies among Pyralidae, namely G. mellonella (Tokarev et al. 2018), and Erebi-
dae, represented by L. dispar (Kononchuk et al. 2021). Susceptible species with high
proportion of specimens displaying developed infection include the type host O.
nubilalis and its congeners (Grushevaya et al. 2020). In the present study, new cases
of susceptible hosts are A. urticae and D. sibiricus. Moreover, in spite of occasional
observation and the limited sample size (N=1), it can also be indicated that E. for-
calis is susceptible to infection with N. pyrausta. Finally, highly susceptible species,
displaying high proportion of insects with developed infection accompanied with
high levels of early mortality, are also found across higher rank taxa. e new cases,
in addition to the previously reported L. sticticalis (Malysh et al. 2021), include A.
io and S. exigua.
From the presented dataset (Table 3), this can be judged that the susceptibility
to a given species of a microsporidian parasite (exemplied here by N. pyrausta)
does not follow a strict high-rank taxonomy-driven pattern in Lepidoptera. Indeed,
susceptible hosts are found in four dierent superfamilies. Moreover, both the re-
fractory and susceptible, as well as highly susceptible species could be found within
a given superfamily (Papilionoidea) and family (Noctuidae).
Table 3. Summary of susceptibility data of lepidopteran insect hosts to Nosema pyrausta
infection under experimental conditions
Insect host Trophic
preferences
Susceptibility
to Nosema
pyrausta*
Reference
Higher-rank
taxonomy
Species Wingspan,
mm
Yponomeutoidea
Yponomeutidae
Yponomeuta
evonymella
16-25 Oligophagous:
Prunus, Sorbus
- is paper
Yponomeuta
malinellus
16-20 Oligophagous:
Malus
- is paper
Plutellidae Plutella
xylostella
15 Oligophagous:
Brassicaceae
- is paper
Ecological vs physiological host specicity: the case of the microsporidium Nosema pyrausta (Paillot) Weiser, 1961309
Insect host Trophic
preferences
Susceptibility
to Nosema
pyrausta*
Reference
Higher-rank
taxonomy
Species Wingspan,
mm
Papilionoidea
Pieridae
Pieris
brassicae
45-60 Oligophagous:
Brassicaceae
- is paper
Pieris rapae 45-50 Oligophagous:
Brassicaceae
- is paper
Pieris napi 45-55 Oligophagous:
Brassicaceae
- is paper
Aporia
crataegi
75 Oligophagous:
Rosaceae, Salix,
Betula
-** is paper
Nymphalidae
Aglais urticae 45-62 Monophagous:
Urtica
+ is paper
Aglais io 50-55 Oligophagous:
Urtica,
Humulus
++ is paper
Junonia coenia 50-65 Oligophagous:
Plantaginaceae
etc.
+ Hall 1952
Pyraloidea
Pyralidae Plodia
interpunctella
18-28 Grain-feeding -** is paper
Galleria
mellonella
30-41 Wax-feeding ± Tokarev et
al. 2018
Crambidae Ostrinia
nubilalis
26-30 Polyphagous,
herbivorous
+ Grushevaya
et al. 2020
Ostrinia
scapulalis
26-30 Polyphagous,
herbivorous
+ Grushevaya
et al. 2020
Ostrinia
furnacalis
26-30 Polyphagous,
herbivorous
+ Grushevaya
et al. 2020
Loxostege
sticticalis
24-29 Polyphagous,
herbivorous
++ Malysh et
al. 2021
Evergestis
forcalis
25-28 Oligophagous:
Brassicaceae
+ is paper
Lasiocampoidea
Lasiocampidae De ndr oli mus
sibiricus
60-120 Oligophagous:
Abies sibirica
Ledebur, 1833,
Larix
+ is paper
Bombycoidea
Bombycidae Bombyx mori 40-50 Monophagous:
Morus
- is paper
Sphingidae Manduca
sexta
95-120 Oligophagous:
Solanaceae
- is paper
310Yuri S. Tokarev et al. / Acta Biologica Sibirica 8: 297–316 (2022)
Our idea that the polyphagous lepidopteran larvae may be more susceptible to
infection with microsporidia as compared to the oligophagous and the monopha-
gous ones, does not nd a conrmation as well. In particular, among polyphagous
herbivorous noctuids, only one out of three tested species turned out to be (highly)
susceptible, while the others two were refractory. On the other hand, monophagous
A. urticae, as well as oligophagous J. coenia, D. sibiricus, and E. forcalis, were sus-
ceptible while oligophagous A. io was highly susceptible.
e size of the insect host species also does not seem to directly dene levels of
larval susceptibility to N. pyrausta, as can be seen from comparison of the small-
est (refractory, P. xylostella) vs one of the largest (highly susceptible, D. sibiricus)
of the tested insects. On the other hand, P. xylostella is known to be a very fast de-
veloping species (Andreeva et al. 2021) while D. sibiricus development is very slow
(Kirichenko et al. 2009) and this might have been a clue to understanding why the
former species is refractory, and the latter is highly susceptible. is assumption,
however, can be opposed by the case of the fast developing L. sticticalis which is
highly susceptible.
Results of microsporidia testing against insects originating from nature should
always be treated with care and veried using appropriate methods of pathogen
species identication to exclude the cases of natural infection, especially in isolates
Insect host Trophic
preferences
Susceptibility
to Nosema
pyrausta*
Reference
Higher-rank
taxonomy
Species Wingspan,
mm
Noctuoidea
Erebidae Hyphantria
cunea
25-42 Polyphagous,
dendrophilic
phyllophagous
- is paper
Lymantria
dispar
37-62 Polyphagous,
dendrophilic
phyllophagous
± Kononchuk
et al. 2021
Noctuidae Helicoverpa
armigera
30-40 Polyphagous,
herbivorous
- is paper
Spodoptera
exigua
26-32 Polyphagous,
herbivorous
++ is paper
Mamestra
brassicae
34-50 Polyphagous,
herbivorous
- is paper
* e insect species is referred to as a) refractory (-) when it develops no infection; b) resistant (±) when
low proportion of insects with developed infection or low intensity of infection is observed; c) susceptible
(+) when high proportion of specimens display developed infection; and d) highly susceptible (++) when
high proportion of insects with developed infection is accompanied with high mortality level.
** e data should be treated as preliminary due to presence of naturally occurring microsporidia which
might have inuenced interactions of the test insects with the experimentally applied pathogen.
Ecological vs physiological host specicity: the case of the microsporidium Nosema pyrausta (Paillot) Weiser, 1961311
and species with similar morphology, as shown for A. crataegi and P. interpunctella.
Presence of a natural infection might have inuenced interactions of the test insects
with the experimentally applied microsporidium. On the one hand, inherent mi-
crosporidia may prevent from infection with an externally applied pathogen species
due to competition. On the other hand, application of a non-specic microsporid-
ium, which cannot infect the insect itself, may however induce activation of other
microsporidia which are present in the covert form (Issi 1986). Anyway, such results
should be considered cautiously and require additional studies using microsporid-
ia-free colonies of the test insects.
In spite of observed infectivity of N. pyrausta to a broad range of Lepidoptera,
it has not been reported in those or similar host species under natural conditions
when extensive surveys were performed. For example, in the Illinois Natural History
Survey (INHS) Collection, none of the 30 microsporidia samples from Crambidae,
Pyralidae, Erebidae, Lasiocampidae, Noctuidae, and other families, showed reliable
identity with N. pyrausta (Tokarev et al. 2020). Similarly, among 161 microsporidia-
positive specimens of S. litura in Japan, none displayed infection with N. pyrausta
(Shigano et al. 2015). Additionally, the survey of the Gebank database indicated
absence of entries which can be identied as N. pyrausta from hosts other than O.
nubilalis. e obtained data are in good agreement with the previous observations
that physiological host range of microsporidia, observed under experimental con-
ditions, is broader than the ecological one, observed in nature (Solter and Maddox
1998). In case of N. pyrausta, one logical explanation is the cryptic lifestyle of the
host larvae, preventing them from excessive exchange of parasite burden with other
Lepidoptera.
Conclusion
Experimental evidence of microsporidian host range is of great importance both
for elucidation of factors which dene insect interactions with eukaryotic micro-
parasites and for evaluation of microsporidia potential for microbial pest manage-
ment, including susceptibility of possible target pest species, suitability of certain
laboratory models for large-scale propagation of the entomopathogens and their
side eects on the non-target entomofauna. In this study, the ability of N. pyrausta
to infect lepidopteran larvae was found to vary depending upon the insect species.
Susceptible hosts are found within dierent taxonomic and ecological groups. High
mortality levels induced in S. exigua suggest N. pyrausta could be a promising agent
to control the pest. Meanwhile, spore yield indices observed in A. urticae indicate
the prospects of in vivo mass production of the pathogen in this host species. By
this research, we encourage other scientic groups to gather as much information as
possible concerning host ranges of microsporidian species they work with.
312Yuri S. Tokarev et al. / Acta Biologica Sibirica 8: 297–316 (2022)
Acknowledgments
Authors are indebted to Yuliya V. Volodartseva and Alsu M. Utkuzova (All-Russian
Institute of Plant Protection, St. Petersburg, Russia) for assistance with stock insect
cultures’ maintenance and to Sergei G. Udalov (ibidem) for capturing insect macro
photographs (Fig. 2). e bioassays using S. exigua could not be possible without
cooperation with Alina Kafel (University of Silesia, Katowice, Poland). e research
was performed using the equipment of the Core Centrum “Innovative Technologies
of Plant Protection” at the All-Russian Institute of Plant Protection and the Core
Centrum "Genomic Technologies, Proteomics and Cell Biology" of the All-Russian
Institute of Agricultural Microbiology (St. Petersburg, Russia). e research was
supported by Russian Science Foundation under grant # 20-66-46009.
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316Yuri S. Tokarev et al. / Acta Biologica Sibirica 8: 297–316 (2022)
Genbank accession numbers:
https://www.ncbi.nlm.nih.gov/nuccore/HM566196
https://www.ncbi.nlm.nih.gov/nuccore/GU828662
https://www.ncbi.nlm.nih.gov/nuccore/KC836092
https://www.ncbi.nlm.nih.gov/nuccore/U26532
https://www.ncbi.nlm.nih.gov/nuccore/FN434087
https://www.ncbi.nlm.nih.gov/nuccore/LC422335
https://www.ncbi.nlm.nih.gov/nuccore/MG456600
https://www.ncbi.nlm.nih.gov/nuccore/D85503
https://www.ncbi.nlm.nih.gov/nuccore/ON256647 (will be made publicly available when
the paper will be published; available at authors by request)
https://www.ncbi.nlm.nih.gov/nuccore/AF426104
