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Journal of Virological Methods
journal homepage: www.elsevier.com/locate/jviromet
Diagnostic protocols for the detection of Acheta domesticus densovirus
(AdDV) in cricket frass
Emilia Semberg
a
, Joachim R. de Miranda
a
, Matthew Low
a
, Anna Jansson
b
, Eva Forsgren
a,⁎
,
Åsa Berggren
a
a
Department of Ecology, Swedish University of Agricultural Sciences, Uppsala 750 07, Sweden
b
Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, 750 07, Sweden
ARTICLE INFO
Keywords:
DNA extraction
Frass
Faeces
Crickets
Acheta domesticus
Densovirus
AdDV
ABSTRACT
The European house cricket (Acheta domesticus) is a species of interest for the emerging insect-as-food industry.
Acheta domesticus densovirus (AdDV) is a member of the Parvoviridae virus family which infects A. domesticus,
causing widespread mortality and even extinction of local cricket populations. Despite the well-known detri-
mental effects of AdDV in commercial rearing of A. domesticus there are no optimized protocols to accurately and
non-destructively detect and quantify the virus. This study establishes a new protocol for the detection of AdDV
in faecal material from A.domesticus. The protocol includes methodological improvements, such as upgrading
from conventional PCR to quantitative real-time PCR and is much more sensitive than previously published
protocols. Moreover, this study shows that cricket faeces are a suitable, non-destructive sample substrate to infer
reliably if a cricket population is infected with AdDV or not. Early detection of lethal or economic threats, such
as disease-causing viruses, is an essential part of commercial cricket management as well as for monitoring the
risk of spread to wild cricket populations or to (human) consumers.
1. Introduction
The European house cricket (Acheta domesticus) is currently used in
insect physiological studies and is reared as feed for pets. It is also a
species of interest for the emerging insect-as-food industry (Clifford and
Woodring, 1990;Szelei et al., 2011;van Huis et al., 2013). Acheta do-
mesticus densovirus (AdDV) is a member of the Parvoviridae virus fa-
mily (Bergoin and Tijssen, 2008;Tijssen et al., 2011;Cotmore and
Davison, 2015) and infects A. domesticus (Styer and Hamm, 1991;Szelei
et al., 2011). The virus can also infect other cricket species, but has only
been shown to be fatal to A. domesticus (Weissman and Gray, 2012).
AdDV infection in cricket populations often results in widespread
mortality and even extinction of local cricket populations (Maciel-
Vergara and Ros, 2017;Szelei et al., 2011). Infected crickets show a
range of symptoms, such as malnutrition, inhibited growth, reduced
fecundity, paralysis and death (Liu et al., 2011;Szelei et al., 2011).
Despite the detrimental effects of AdDV to wild A. domesticus popula-
tions and for the commercial rearing of the species, there are no well-
developed and optimized protocols to accurately and non-destructively
detect and quantify the virus. Such a tool would be invaluable for
densovirus epidemiological studies and surveillance, which are
essential for the sustainable rearing of A. domesticus, especially if this
species will be mass reared for human consumption or as feed for fish
and livestock (Berggren et al., 2018;Jansson and Berggren, 2015;van
Huis et al., 2013). Since AdDV is spread through oral-fecal transmission
(Szelei et al., 2011), cricket frass (faeces) is a promising sample type for
non-destructive viral screening. Individual frass samples can also be
used to determine virus prevalence in populations, and can easily be
pooled for population-level analyses. A sensitive frass-based virus de-
tection method would make it possible to detect infection in a cricket
rearing facility at an early stage before clinical symptoms emerge, po-
tentially minimizing disease spread between sub-populations. Thus, a
non-destructive screening protocol for densovirus in cricket populations
would be a major development in improving cricket rearing standards.
The aim of this study was therefore to develop and optimize a quanti-
tative assay for AdDV detection in cricket frass and to develop this into
a sensitive, accurate and reproducible screening protocol.
https://doi.org/10.1016/j.jviromet.2018.12.003
Received 10 September 2018; Received in revised form 14 November 2018; Accepted 1 December 2018
⁎
Corresponding author.
E-mail address: eva.forsgren@slu.se (E. Forsgren).
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2. Material and methods
2.1. Origin of samples
Acheta domesticus used in this study were collected from a wild
cricket population (hereafter ‘wild’) outside Uppsala, Sweden and a
commercially-reared cricket population (hereafter ‘reared’), bought in a
pet shop in Uppsala. The wild and reared crickets were quarantined
from each other in isolated cages, and each of these groups was further
divided into two separate cages. Frass samples were collected from the
bottom of each of the cages after 24 h and 1 week and stored in col-
lection tubes at −20 °C until processed.
2.2. DNA extraction from crickets and frass
AdDV was confirmed to be present in the reared crickets, by as-
saying two dead and one live specimen according to the protocol of
Szelei et al. (2011), with the following modifications: individual
crickets were placed in a Bioreba mesh bag (Bioreba, Reinach, Swit-
zerland), flash frozen with liquid nitrogen and ground to a powder
using a pestle. The resulting powder was mixed with 2 mL nuclease-free
water and further homogenized by centrifuging through a QIAshredder
(Qiagen, Hilden, Germany). One hundred μL cricket homogenate was
mixed with 180 μL Buffer ATL and 20 μL proteinase K (Qiagen, Hilden,
Germany) and incubated at 56 °C for 3 h. DNA was purified from the
homogenate by a QIAcube extraction robot (Qiagen, Hombrechtikon,
Switzerland) following the Qiagen DNA extraction protocol for Tissues
and Rodent tails, eluting in 200 μL AE buffer (Qiagen, Hilden, Ger-
many). The DNA concentration was estimated using a NanoDrop 1000
instrument (NanoDrop, USA) and stored in −20 °C until further use.
The DNA extraction protocol for frass was adapted from the pub-
lished protocol for purifying cricket DNA (Szelei et al., 2011) as follows:
0.1 g frass was homogenized in 0.5 mL nuclease-free water in a Mix-
erMill 400 (Retsch Haan, Germany) at maximum speed (30 f/s) for
1 min using 10 glass beads (ɵ3 mm). One hundred μL homogenate was
mixed with 180 μL ATL buffer and 20 μL proteinase K (Qiagen, Hilden,
Germany), incubated at 56 °C for 1–5 h and extracted using a QIAcube
extraction robot (Qiagen, Hombrechtikon, Switzerland) following the
Qiagen DNA extraction protocol for Tissues and Rodent tails, eluting in
200 μL buffer AE (Qiagen, Hilden, Germany). The DNA concentration
was estimated using a NanoDrop 1000 instrument (NanoDrop, USA),
diluted to a final concentration of 10 ng/μL and stored at −20 °C until
further use.
2.3. Real-time quantitative PCR
The quantitative real-time PCR (qPCR) assays for AdDV detection
were designed around the primers previously described for amplifying a
305 bp fragment (VP) located in the virus capsid protein gene cassette
and a 357 bp fragment (NS) located in the non-structural region (Szelei
et al., 2011; Supplementary Table I). A constant amount of template
DNA was included in each reaction, to minimize template concentra-
tion-dependent bias in qPCR efficiency (Nolan et al., 2007;Forsgren
et al., 2017). The qPCR reactions were run using the EvaGreen®SYBR
Green kit (Bio-Rad, Singapore) containing 0.4 μL of each primer (10
μM) and 2 μL template DNA (20 ng) in 10 μL total volume. The qPCR
reactions were run in a CFX-Connect thermal cycler (Bio-Rad), with
following cycling conditions: initial enzyme activation step at 98 °C for
2 min followed by 40 cycles of denaturation at 98 °C for 10 s and an-
nealing/extension at 58 °C (AdVP-primers) or 62 °C (AdNS-primers) for
30 s. The amplification was followed by a melting curve analysis
starting with 65 °C for 5s with 0.5 °C increments up to 95 °C. All assays
were run in duplicate if not stated otherwise.
2.3.1. qPCR performance parameters
Each reaction plate contained positive and negative (non-template)
assay controls. For each assay, a quantitative calibration curve was
established through a 10-fold dilution series of a positive control
(purified PCR product) of known concentration, covering 6 orders of
magnitude. These positive controls were used for quantitative data
conversion, establishing the reference melting curve profile of the
amplicon and for estimating the qPCR performance statistics.
2.3.2. Annealing temperature optimization
The annealing-extension temperature was optimized for product
specificity and detection sensitivity using a 55–69 °C temperature gra-
dient for both assays, followed by narrower separate gradient intervals
for each assay between 59–63 °C (AdNS) and 56–61 °C (AdVP), based on
the data from the first gradient.
2.4. DNA extraction optimization
2.4.1. Optimization of Proteinase-K incubation
Three different proteinase-K incubation times (1, 3 and 5 h) were
tested (n = 12 for each). This was done to establish the minimum ne-
cessary incubation time for proteinase K, with the minimum time being
judged as the time beyond which no significant increase in PCR copies
were detected, in either of the qPCR assays (AdNS and AdVP).
2.4.2. Evaluation of a post-homogenization centrifugation step
Frass homogenate is very thick and difficult to pipet, which can
affect pipetting accuracy, and thus the variability of the assay. A test
was therefore conducted to determine if centrifuging the homogenate at
8000gfor 1 min and analyzing the supernatant instead of the whole
homogenate would improve the robustness of the protocol without
sacrificing sensitivity. Preliminary results (using the initial protocols of
Szelei et al., 2011) showed that the reared crickets contained high le-
vels of AdDV, whereas the wild crickets appeared to be free of AdDV.
Four frass samples were prepared as follows: samples R1 and R2 came
from 2 separate cages containing AdDV-infected reared crickets; sample
W1 came from a cage containing wild crickets (determined previously
to be AdDV-free); and sample R1-W1 consisted of 14% R1 frass and
86% W1 frass, in order to create a sample with intermediate AdDV
levels (see Supplementary Table II). A single homogenate was prepared
from each frass sample. The frass was weighed, 5 u L nuclease-free
water was added per mg frass and the mixture was homogenized with a
MixerMill 400 (Retsch Haan, Germany) and 10 glass beads as described
above. This primary homogenate was split into 2 equal duplicate test
homogenates. From each duplicate test homogenate, 100 μL was re-
moved for direct DNA extraction while the remainder was centrifuged
at 8000gfor 1 min, after which 100 μL supernatant was removed for
DNA extraction, as described above.
2.5. Limits of detection, LOD
2.5.1. LOD for the qPCR assay
The limit of detection (LOD) of the qPCR assays was determined
through two replicate 10-fold serial dilution series of an AdDV-positive
frass DNA sample with a 10 ng/μL starting concentration. Each assay
was run 8 times at each dilution level, for both dilution series. The LOD
is defined here as the estimated amount of target DNA, as determined
by qPCR, at the highest dilution level where the target was detected by
all 16 replicate reaction assays in both dilution series.
2.5.2. LOD for the entire protocol
The entire protocol LOD was tested similarly, but starting with a
crude virus-positive frass homogenate diluted with virus-free frass
homogenate through a 10-fold dilution series. Three independent
homogenate dilution series replicates were prepared from the same
original homogenates. The diluted frass homogenates were extracted as
described above, the DNA diluted to 10 ng/uL and each qPCR assay was
run in duplicate on each template, using reaction conditions and
E. Semberg et al. -RXUQDORI9LURORJLFDO0HWKRGV²
annealing temperature-optimized thermos-cycling profiles described
above.
2.6. Statistical analyses
To check for differences between assay types, incubation times and
to account for assay replicate series generalized linear mixed models
(GLMM) were used including the ‘lmer’function from the R-package
‘lme4’(Bates et al., 2015). For the different proteinase K incubation
times the number of genome equivalents were compared, based on an
interaction between the assay type (AdNS and AdVP) and the incuba-
tion time (as a 3-level categorical variable), while controlling for in-
cubation and extraction repeats as random variables. To output pre-
dictions from these mixed models, 1000 simulations were bootstrapped
for each incubation time and each assay using the ‘ezPredict’function
from the R-package ‘ez’(Lawrence, 2016). For the limit of detection
(LOD) estimates, the number of genome equivalents from the two as-
says were compared, using the data from the final dilution level that
successfully detected virus in all replicates. For this a GLMM was used
that included the replicate dilution series as a random variable. Con-
sistency between replicate dilution series for the different assays was
examined by using data from all dilutions and comparing replicate
series (with series as a fixed effect and dilution level as the random
effect in the mixed models).
3. Results
3.1. Real-time quantitative PCR
3.1.1. qPCR performance parameters
Both the AdNS and AdVP assays displayed near-perfect inverse-
linear relationships between the Cq-value and log10 [template] across
nine orders of magnitude (R2 = 0.999 for both assays), with excellent
PCR reaction efficiencies (94.5% and 93.6% respectively for the AdNS
and AdVP assays) as calculated from the respective slopes of these re-
lationships (Supplementary Table I).
3.1.2. Annealing temperature optimization
The qPCR annealing temperature optimization experiments showed
that the optimal annealing temperature was 62 °C for the AdNS assay
and 58 °C for the AdVP assay, these being the highest annealing tem-
peratures to generate PCR product without compromising the assays’
qPCR performance parameters (Supplementary Table I).
3.2. DNA extraction optimization
3.2.1. Proteinase K incubation
The extraction tests with proteinase K showed that a 1 h incubation
time was appropriate, with no increase in assay detection sensitivity
after this period (Supplementary Fig. 1). Although the baseline sensi-
tivity differed between the assays (t = 14.7, P < 0.001), the lack of
increase in detection sensitivity with increasing incubation times was
the same for both assays (assay*time (3 h) interaction: t = 0.54;
P = 0.58 & assay*time (5 h): t = 0.66; P = 0.51; see Supplementary
Figure 1).
3.2.2. Homogenate centrifugation step
Including the homogenate centrifugation step in the protocol and
analyzing the supernatant instead of the whole homogenate improved
the AdDV detection sensitivity by 1.71x to 5.69x (Table 1).
3.3. Limits of detection, LOD
3.3.1. LOD for the qPCR assay
The limit of detection (LOD) for the qPCR assays was at the 1/1000
dilution level from the baseline (Supplementary Table III).
3.3.2. LOD for the entire protocol
The LOD was also determined for the entire detection protocol,
based on successively diluting the virus-contaminated homogenate with
virus-free homogenate. Through to the 1/10000 dilution level all re-
plicate extractions and assays had a 100% detection rate, with an es-
timated 3.37 ± 1.13 AdDV genome equivalents detected by the AdVP
assay, and 1.82 ± 1.07 AdDNA genome equivalents by the AdNS
assay. There was no statistically significant difference between the re-
plicate homogenate dilution series, for either of the qPCR assays (AdVP:
F2, 40 = 1.15, P = 0.33; & AdNS: F2, 40 = 1.07, P = 0.35). At all di-
lution levels, the AdVP assay consistently detected higher levels of
AdDV than the AdNS assay (n = 30 for each assay; t = 3.8, P < 0.001;
Supplementary Table IV).
4. Discussion
For general health screening of animal populations, it is essential to
have a reliable, fast and preferably non-destructive screening and as-
saying protocol. This is particularly important for intensively reared
animals in production facilities where there is a high risk of damaging
disease outbreaks. A reliable screening protocol is moreover essential
for good animal husbandry and hygiene, so as to minimize production
losses, improve animal welfare, protect susceptible wild animal popu-
lations from disease and to minimize the risk of contaminating human
food or animal feed products (Berggren et al., 2018).
With this study a basic protocol has been established for the analysis
of faecal material from the domestic cricket (A.domesticus) as a suitable
sample type for the detection of AdDV, a lethal virus disease of crickets
that is particularly prevalent and damaging in highly intensive cricket
rearing facilities. Previous protocols have been developed for the ana-
lysis and screening of body parts of crickets (Weissman and Gray,
2012). The main purpose of this study was to establish and optimize
principal parameters for a non-invasive diagnostic protocol for AdDV
detection based on cricket faeces. The resulting protocol has been de-
veloped from a previously published protocol for the qualitative PCR-
based detection of AdDV in whole crickets (Szelei et al., 2011). The
principal improvements are: the upgrading of the PCR protocol to real-
time quantitative detection (qPCR) using the EvaGreen dye-based de-
tection system, which results in much lower limits of detection (LOD)
for both the individual qPCR assays and the entire diagnostic protocol
than those published previously (Szelei et al., 2011;Weissman and
Gray, 2012); the inclusion of a low-speed centrifugation step after frass
homogenization to clarify the extract, which facilitates sample man-
agement and improves detection sensitivity, and the reduction of the
proteinase-K incubation step to one hour without loss of sensitivity.
Minor optimizations were made to the annealing temperatures of the
qPCR assays themselves, which were optimized at 62oC for the AdNS
assay and 58oC for the AdVP assay. There was no significant difference
in detection sensitivity or assay performance between the two qPCR
assays employed, which are based on different regions of the AdDV
genome. The increased sensitivity of the protocol through the inclusion
of a clarification step shows that the virus is mostly contained within
the soluble fraction for the frass. The improved detection sensitivity is
mostly likely through the enrichment of the extracted DNA with AdDV
DNA, by pelleting extraneous faecal material.
It has previously been suggested that AdDV is extremely resistant to
proteinase-K digestion (Weissman and Gray, 2012;Tijssen et al., 1977).
No difference was found in AdDV detection sensitivity between the 1, 3
and 5 h proteinase-K incubation periods. There was no difference be-
tween the two qPCR assays throughout this experiment, implying that
the two genomic regions where the assays are located were equally
affected by the proteinase-K treatments. Either the proteinase-K diges-
tion was ineffective throughout (Weissman and Gray, 2012;Tijssen
et al., 1977), in which case only non-packaged AdDV DNA was de-
tected, or highly effective, such that all relevant proteinase digestion
was completed after 1 h.
E. Semberg et al. -RXUQDORI9LURORJLFDO0HWKRGV²
This study shows that it is possible to use A. domesticus frass samples
to determine if (reared) cricket populations are infected with Acheta
domesticus densovirus (AdDV). The protocol can contribute to mini-
mizing the risk and effects of densovirus outbreaks in cricket rearing
facilities, improve animal welfare (Gjerris et al., 2016) both through
improved disease management and non-destructive sampling, and can
be a valuable tool for improved management of commercial cricket
rearing facilities. The protocol can be developed further and areas for
improvement will arise with its use, such as for example the relation-
ship between bulk frass-based AdDV detection rates and levels and the
proportion of infected individuals this represents. The answer to this is
partly due to the interaction between the virus and cricket and the
resulting behavioral and physiological responses, but further develop-
ment of the method will bring light to this important area.
Conflicts of interest
The authors declare no conflict of interest. The funding sponsors
had no role in the design of the study; in the collection, analyses, or
interpretation of data; in the writing of the manuscript, or in the de-
cision to publish the results. Original data is available on request to the
corresponding author.
Author contributions
ES, EF & JM conceived and designed experiments; ES performed
experiments; ES, ML & ÅB analysed the data; AJ, ML & ÅB contributed
reagents/materials/analysis tools; ES, EF, JM, ML & ÅB wrote the
paper. All authors read and approved the paper.
Acknowledgements
This project was financed by grant 2016-00361 from the Swedish
Agricultural Research Council (FORMAS). We would like to thank Peter
Tijssen and Judit Pénzes from the INRS-Institut Armand-Frappier in
Canada for sharing a plasmid positive control for the AdVP assay.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jviromet.2018.12.003.
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Table 1
Starting quantity (SQ), the variation in SQ-values ( ± s.d.) and the proportional difference (SQSup/SQHom) between assays based on whole frass homogenate
(Homogenate) or the post-centrifugation supernatant of the frass homogenate (Supernatant). All values have been divided by 10
6
for ease of presentation.
AdNS AdVP
Sample SQ ± s.d Homogenate SQ ± s.d Supernatant SQ
Sup
/SQ
Hom
SQ ± s.d Homogenate SQ ± s.d Supernatant SQ
Sup
/SQ
Hom
R1 61.90 ± 0.43 112.00 ± 19.4 1.81x 73.00 ± 6.07 125.00 ± 39.6 1.71x
R2 3.20 ± 0.77 9.50 ± 3.84 2.88x 4.13 ± 0.884 8.63 ± 3.63 2.09x
R1+W1 3.00 ± 3.00 17.10 ± 0.51 5.69x 5.03 ± 0.055 18.40 ± 1.74 3.65x
W1 0 0 0 0 0 0
E. Semberg et al. -RXUQDORI9LURORJLFDO0HWKRGV²
