Cerere De Finantare Section Fin.pdf [621712]

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PN-III-CERC -CO-PED -2-2019

FUNDING APPLICATION

Improved thelytokous parasitoids to be used against synanthropic fly pests in organic animal farms

IMPASY
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PN-III-CERC -CO-PED -2-2019 B. Project Proposal

B.2 Scien tific description (will be uploaded into the platform)

B. 2.1 Project Scope and Objectives
The content of this section corresponds to the first evaluation criterion (project scope and objectives).

Project scope and description of the model system.
Synan thropic flies are insect pests related to major human activities such as food processing and animal
husbandry. They include among others the house fly Musca domestica L. and the common stable fly Stomoxys
calcitrans (L.) that cause major health proble ms to both humans and livestock: nuisance, pain, weight loss due to
avoidance behaviour (“fly worry” syndrome), transmission of pathogens etc. (e.g. Ballesteros et al. 2011). Their
control is problematic, being traditionally based on chemical insecticides that also have toxic effects in humans.
Additionally, the flies can develop resistance to insecticides in a short time leading to the failure of many vector
control campaigns since long ago (Keiding 1986). House flies are resistant to virtually every insec ticide used
against them (Scott et al. 2000 , Meisel & Scott 2018, Freeman et al. 2019 ). An alternative to chemical control is
the biological control , which uses the natural enemies of the pest. Most natural enemies of the synanthropic flies
are small parasitoid wasps (Insecta: Hymenoptera) that develop as larvae in flies’ puparia killing them in the
process (e.g. Skovgård & Nachman 2004). Because of the low parasitism levels and low dispersal (Machtinger,
Geden & Leppla 2015 b), fly control programs are based on m ass releases of laboratory reared wasps (Stafford
2008) and this project intends to develop the technology to produce locally adapted and improved
thelytokous lines of parasitoid wasps . The main potential beneficiary of this technology is the org anic
farmi ng—especially poultry and dairy —since biocontrol is a main component of this agricultural system that
aims at producing crops and livestock with minimal harm to the environment, animals or humans (Romania uses
the equivalent term ecological agric ulture; EC C No. 834/2007). The last available report shows that the total
organic area of EU -28 was 12.6 million ha in 201 7 (the most recent available data) and continuing an upward
trend (Anonymous 201 9). According to MADR (Ministerul Agriculturii si Dezv oltarii Regionale) , the total
organic area of Romania in 2018 was 326 259,5 ha. Arable land crops predominate but are closely followed by
pastures and meadows (used for grazing organic livestock) accounting cca 2 0%. Additionally, and importantly,
in 2014 Romania rec orded one of the highest increase in the number of organically farmed sheep (+43%) and
cattle (+68%) in the whole EU -28, currently Rom ania has 10 176 400 organically farmed sheep s and 1 977 200
organically farmed cows (Anonymous 201 9). In poultry, livestoc k and dairy organic farming of outmost
importance is the prevention of diseases with a good diet and keeping the sheds clean. In such a system
pestiferous flies can be controlled with parasitoid wasps . A successful biocontrol program must satisfy two
prere quisites. (1) The accurate identification of parasitoid species and their host associations are necessary pre –
requisites for improving pest control and to limit any unintended deleterious effects. This first step was
addressed in one of our past research p roject (PARASYN – PNII -RU-TE-2012 -3-0057; 2013 -2016) that
involved most of the team members of this proposal. We developed standard molecular markers that will be
further used for this proposal and showed the unexpected presence of m olecularly distinct cry ptic species. (2)
The second major obstacle in using parasitoids in biocontrol is the difficultly of having adequate lines of
parasitoids that are (a) naturally adapted to local conditions, (b) unable to hybridize with closely related
species or other popu lations thus losing their genetic identity, and (c) consist mainly in females (males are a
“burden” for the mass production, as they do not parasitize hosts).
A solution to the above -mentioned problems is to have a thelytokous line of female parasitoids . In
Hymenoptera, thelytoky is a rarer form of parthenogenesis in which females produce exclusively females from
unfertilized eggs (Gottlieb et al. 2002). Thus, a thelytokous female will not need males to reproduce and once
released outside the laboratory wil l not hybridize (maintaining a pure line) and the numbers of obtained
individuals (i. e. the commercial product) will be maximized, as no resources are used to rear males. All
individuals relea sed for biocontrol (females only) will be capable of destroying flies, otherwise about 50% are
males that do not contribute directly to the control of the pest (Aeschlimann, 1990). Also, parthenogenetic
species represented by females only establish quickly locally adapted populations after introduction in
agricultural systems (Hoffmann et al . 2008). From the common fly parasitoids only Muscidifurax uniraptor
Kogan & Legner is thelytokous due to a Wolbachia infection (Gottlieb et al. 2002) and importantly, there are no

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PN-III-CERC -CO-PED -2-2019 deleterious physiological effects (Zchori -Fein et al. 2000). However, Muscidifurax wasps have a lower
parasitism (fly destruction) rates compared to Spalangia wasps (e.g. Gibson & Float 2004) . They also tend to
search for hosts (fly puparia) on the surface, while Spalangia searches at greater depths (Johns on et al. 2017) . As
there are no naturally thelytokous Spalangia or Trichopria known , they would need to be produced
experimentally , this being the main goal of this project . Thelytoky can be induced by infecting females with
Wolbachia or other endosymbion ts (Cardinium and Rickettsia , but for simplicity Wolbachia is used throughout
the text). Wolbachia are members of the order Rickettsiales: parasitic, mutualistic and commensal intracellular
bacteria. They are maternally inherited within the host lineage an d do not spread as an infection (Werren et al.
2008). A recent review suggest that they might be present in more than 65% of all arthropods species
(Hilgenboecke r et al. 2008) including parasitic wasps (e.g. Gottlieb et al. 2002; Floate, Coghlin & Taylor 2 008).
It was recently estimated that a bout 99% of the diversity of Wobachia is unknown (Detcharoen et al. 2019).
For our experiments we intend to use species of parasitoids from the genera Spalangia and Trichopria , for the
following reasons: (a) The two ge nera are among the most successful natural enemies of synanthropic flies. (b)
They have contrasting life histories: Spalangia are solitary parasitoids ( only one individual develops per fly
pupa rium ) and Trichopria are gregarious (several individuals develo p in a single pupa rium ), and thus we can test
the experimental procedures in two different situations. (c) In Europe there are no biological control products
against synanthropic flies on the market. (d) We have extensive taxonomic expertise in thes e taxa, as our
published results show. (e) We successfully finished a research project that aimed at a better identification
system of these parasitoids worldwide using standardized molecular markers, and discovery of new cryptic
species with potential use in bio control. The results of this project can now be used one step further.
To summarize , the final objective of the project is to create lines of thelytokous females of Spalangia and
Trichopria by using Wolbachia endosymbionts. This would represent a first st ep towards the commercial use
of these “improved” parasitoids against synanthropic fly pests in ecological animal farms.

Novelty and relevance o f our p reliminary results.
The present proposal intends to ma ke the best of the resources generated by the proj ect “ Parasitoids of
synanthropic flies: advanced identification methods using an integrated approach ” (project leader Mircea D.
Mitroiu, responsible for the molecular activities Lucian Fusu; PARASYN – PNII -RU-TE-2012 -3-0057)
(https://sites.google.com/site/parasyn2013/home ): over 50 species and 500 specimens of parasitoids of flies, 150
DNA sequences of the mitochondrial CO 1 gene (DNA barcode sequences), 100 DNA sequences of the nuclear
genes 28 S and 18S (rRNA). Out of 64 species of Spalangia only 9 have barcode sequences available on NCBI.
Though there are 51 published sequences of the 18S rRNA gene and 42 sequences for the 28S rRNA gene most
are derived from only one species (Taylor et al. 2006 ; Munro et al. 2011; Kohei & Ryo 2009). Additionally ,
there are 30 sequences for the 12S rRNA gene (Gila di et al . 2000), 9 sequences of protein coding genes
(Desjardins et al. 2007; Martinson et al. 2016; Dewaard unpublished; Krogmann et al. unpublished) and one
transcriptome shotgun assembly (Peters et al. 2018), are available, as well. At this moment the genus Trichopria
comprises a number of 65 species. Though there are 94 CO1 sequ ences on NCBI they are from only 4 identified
and one unidentified speci es (Hebert et al. 2016; Kacar, unpublished). There is also a transcriptome shotgun
assembly for Trichopria drosophilae (Peters et al. 2017).
In Europe there are no commercially availa ble biocontrol products against synanthropic flies: two of the
largest p roducers of biocontrol agents on the European market, Biobest Belgium ( http://www.biobestgroup.com )
and Biotop France ( http://www.biotop.fr ), do not offer any such products. In the United States of America
parasitoid wasps in the family Pteromalidae are largely commercially available: Muscidifurax spp., Spalangia
spp., and Nasonia vitrip ennis (Walker). Trichopria nigra (Nees) (Diapriidae) is researched for the same purpose
in the United States (Ferrero 2008) . Our team recently investigated a line of T. sociablis Masner and found
promising , reaching a 50% parasitism rate (Postu, Popovici & Mitroiu 2013). Also, the research on the
hymenopteran parasitoids of synanthropic flies is evidently more intense involving numerous research groups in
the North America ( Gibson & Floate 2004 , Machtinger et al. 2015 ab, Tayor et al. 201 6, Johnson et al. 2017 ,
Burgess et al. 2017, King et al. 2018, etc.) compared to Europe ( ex. Skovgård & Nachman 2004, de Pedro et al.
2019). With the current European regulations for the import of exotic species to be used as biological control
agents it would be very difficult to import the species from the United States for example , even if they are
efficient . Having European species available for this purpose would greatly facilitate their adoption as biological

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PN-III-CERC -CO-PED -2-2019 control agents on the European market . Especially if it will be shown by us that the used species are genetically
homogenous across Eur ope and do not represent an assemblage of cryptic lineages.

Objectives .
All our objectives, including the intermediate ones , are:
(1) to test alternative artificial media to grow fly larvae in laboratory and to identify the most affordable
one that uses readily available ingredients;
(2) to establish and propagate one to several lines of parasitoid wasps of synanthropic flies, derived fro m
local populations and adapted to the climatic conditions from Romania (these lines are already a potential
commercia l product, even without the improvement from the next objective);
(3) to test the candidate species for the presence of endosymbionts and cure any innate infection
(4) to obtain lines of thelytokous females by artificially infecting them with thelytoky -inducing
Wolbachia endosymbionts and characterize them using genetic markers (high risk -high reward objective) ;
(5) Because these lines ar e a novel product, and not simply wild -caught specimens, they can be patented.
Hence our final goal is to obtain pate nts for these lines that can be exploited commercially. In the next step
we plan to apply for a spin -off or start -up grant. We hold all th e necessary prerequisites to proceed from present
stage to final product. We have access to a large database of parasi toids of synanthropic flies that was generated
by our team in an anterior research project and standardized molecular markers (see above), we are experienced
in rearing parasitoids of flies and other insects, and we have the necessary knowledge and skills to obtain lines of
wasps with horizontally transferred Wolbachia .

Current state of the TRL (technology readiness level) and the intended level.
TRL level 1 – basic principles observed – was completed during the PARASYN project. As detailed above
we have a good understanding of the system, having detected several cases of cryptic species with distinct
climatic preferences. We also have the molecular tools to differentiate most Spalangia and Trichopria species,
an important prereq uisite in their monitoring. Currently , we are at TRL level 2 having a technology concept
formulated . As detailed above, our concept is that t helytokous lines with high specificity for the intended host
will be much more effective, and furthermore, there is no risk that t hey will suff er after release a loss of the
desired traits through hybridization and introgression with the wild populations.
Starting from here we intend to reach by the end of the project TRL level 4 , i.e. to have a technology
validated in the lab. To get to this le vel we will develop our project based on (1) techniques of rearing fly
maggots (e.g. Hogsette 1992) and parasitic wasps ; (2) establish the infection status in the w ild populations of
parasitoid wasps using cloning and sequencing of the 16S rRNA or next generation sequencing (e.g. Ca rpi et al.
2011); (3) cure with antibiotics the original Wolbachia (or other endosymbionts) infection in the wasps (e.g.
Zchori -Fein et al. 2000; Giorgini, 2001); (4) transinfect the females with a strain of Wolbachia known to induce
thelytoky in other species (obtained from dissected ovaries of the donor species by microdissection —see Fusu
(2008 ) or Popovici & Johnson (2012 )—and propagated in an insect cell culture ); (5) screen the progeny of these
female s and determine the Wolbachia titer with qPCR; (6) further maintain in culture and select the lines with
the highest Wolbachia titer that might eventually lead to thelytokous lines ; and (7) besides the already available
CO1 marker to add an ITS marker and a Wolbachia gene marker, to have a quality control tool to identify the
parasitoid wasps at line level (a “unique signature for each line) . Well characterized t helytokous lines with
high specificity for the host are our final goal.

B.2.2 Presentation of the concept of technology / product or existing model which constitutes the starting
point of the project
The content of this section corresponds to the second evaluation criterion (presentation of the concept of
technology/product) .

Preliminary results available prior to the project application.
Our preliminary results from the project PARASYN (PNII -RU-TE-2012 -3-0057 , 2013 -2016 )
(https://sites.google.com/site/parasyn2013/home ) is a database of over 500 specimens of parasitoids of flies with
associated distribution , host data, DNA barcode sequences, and sequences of two nuclear ribosomal RNA genes .
The data generated by the project are uploaded in public databases: MorphBank for the high resol ution images

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PN-III-CERC -CO-PED -2-2019 and GenBank (DNA sequences) from where they will be automatically uploaded in BOLD (DNA sequences).
All this wealth of data is not yet released fo r the public access, awaiting the publication of a paper in a major
journal , but before this data become public, we wish to capitalize on them for the current grant proposal . These
data indicate the presence of cryptic species with apparently discordant ec ological preferences within at least two
species of European Spalangia with potential for use in biological control: S. nigripes and S. fuscipes (Fusu et al.
2014; Mitroiu et al. 2013a, 2014) and discrepancies between the species groups delimi ted based on morphology
(Gibson 2009) and those on molecular data (Fusu et al . 2015a , b). Solving these discrepancies is essential
because they lower the predictive value of a classification. Phylogenetic classification systems that underline the
true evolutionary process can be used to pr edict for example the biology/host preferences of a species if these
characteristics are known fo r the evolutionary close species. Knowing about the existence of cryptic species is
also important for several practical reasons: in the above example about S. nigripes and S. fuscipes in each
species pair one has a southern distribution, being potentially useful for biocontrol during the summer months,
the other has a central and northern distribution being adequate for spring and autumn. We also have extensive
data on t he species of parasitoids of synanthropic flies occurring in Romania (Mitroiu 2013), this being t he
starting point for this project. Our study also showed that the CO 1 barcode sequence is variable enough to be
used as a molecular maker to differentiate spe cies. We will combine the CO 1 sequences with the ITS1 sequence
that is more variable plus the Wolbachia sequence to further distinguish the lines/strains of parasitoid wasps.
These three sequences will provide a unique “signature” of each strain that can b e used to recognize it in field
settings and differentiate it from conspecific wild individuals.
For Trichopria presently we have data on reproductive behavior, a good rearing protocol, and we know it is
a very good candidate for a locally adapted line that can be further improved by infection with Wolbachia in an
attempt to make it thelytokous. Our strain of T. sociabilis is local, being collected in Romania near Iasi, it has a
24% parasitism rate in the field and 50% in confined laboratory conditions (potentially similarly high i n confined
conditions in shades and stables), and most importantly, the sex ratio is already fema le biased (Postu, Popovici &
Mitroiu 2013). Trichopria females have preference for the host they were reared upon in the laboratory (native
host) as the result s of the PARASYN project and the research of Ferrero (2008) show. This gives the opportunity
to have mostly host -specific parasitoids for different target fly species, depending on the native host.
During the PARASYN project we collect ed and establish ed (Mitroiu et al. 2013 b) captive populations of
Spalangia and Trichopria (more species will be collected in the first stage of this project). These species can be
used and improved within this proposal.
Another connected project (PI was director) is a b ilateral project for cooperation Romania -France, Brîncuși –
Hubert Curien Partnership , on the integrative taxonomy of parasit oids of pest insects (IntegPar, 2017 -2018).
During the visits to France at Sophia Agrobiotech 4 members of our team acquired expertise on rearing methods
of pest insects and their parasitoids. Another ongoing project (NEVPIT, PN -III-P4-ID-PCE-2016 -0233, 20 17–
2019, director M.D. Mitroiu) is on a related theme (the integrated taxonomy of the parasitoids of the invasive
green vegetable), bu t it enabled us to gain expertise in laboratory rearings of insects and on collecting with
sentinel baits (eggs in this ca se).

Team members and their expertise .
Coordinating institution (UAIC)
Reader Dr Lucian Fusu (PI): PhD in Biology (Entomology) (2009). Expertise in the taxonomy of parasitic
wasps (since 2010 Fauna Europaea specialist for Chacidoidea) ; cryptic species di scovery using molecular
markers and ecological preferences analysis ( ex. Al khatib et al. 2014, 2016, Gibson & Fusu 2016 , Fusu 2017 );
masters DNA manipulation and phylogenetic analysis techniques (e.g. Al khatib et al. 2014, 2016 ; Fusu 2017) ,
good knowledg e of NGS (Cruaud et al . 2019), knowledge of animal cell culture (currently teaches a Master
course on Animal cell cultures). Published one book (monograph, Magnolia press, Auckland), 4 book chapters,
24 papers on parasitic Hymenoptera (3 8 papers in total), including 24 (18 as principal author) in international
peer-review journals with impact factor . Of these 3 are in the first quarter -Q1 of the journal ranking list of the
discipline (“red” category, one as sole and principal author) and four in the second quarter -Q2 for that
publicatio n year (“yellow” category, three as unique /principal author, one as third author). Team member
(experienced researcher) in the project PARASYN on the parasitoids of synanthropic flies: responsible for the
molecular work; devel oped, organized and is managin g an insect molecular taxonomy laboratory. Director of a
bilateral project for cooperation Romania -France, Brîncuși -Hubert Curien Partnership : integrative taxonomy of

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PN-III-CERC -CO-PED -2-2019 parasitoids of pest insects. During the visits in France to Sophia Agrobiotech acquired expertise on rearing
methods of pest insects and their parasitoids. Further exp erience in leading grant s by being beneficiary or
director of other 9 smaller grants ( 5 SYNTHESYS grants provided by EU funding through FP7, a Percy Sladen
Memorial Fund grant provided by the Linnean Society in London , a postdoctoral fellowship, one intern al grant
and one MC grant ). Role : overseeing all the activities, field collecting, testin g for innate or artificial Wolbachia
infections (PCR, qPCR, TA cloning/NGS), creation of Wolbachia -infected Drosophila cell lines,
characterization of the lines using molecular markers. CV available here .
Reader Dr habil . Mircea D. Mitroiu (researcher) : PhD in Biology (Entomology) (2006) ; habilitation to
supervise PhD students (20 16). Expertise in the taxonomy of Pteromalidae. Published 9 books/book chapters, 52
papers on parasitic Hymenoptera including 1 8 (most as principal author ) in international peer -review journals
with impact factor (Zootaxa , J Hym Res , etc.), 2 on the “yello w” CNCSIS category ( Eur. J. Taxon., Ent. Sci. )
and one in PLoS ONE (Q1, “red”) . Since 2010 Fauna Europaea Group Coordinator for Hymenoptera -Apocrita
(http://www.faunaeur.org/experts.php?id=662 ). Di rector of the project PARASYN and NEVPIT . Role : field
collecting, identifying and rearing Spalangia , curing the innate infections with antibiotics, maintaining
Spalangia lines.
Lect. Dr Ovidiu A. Popovici (researcher) : PhD in Biology (Entomology) (2007). E merging as the leading
expert on the European scelionid wasps (Platygastridae) and other Proctotrupoidea s.l. 36 published papers on
parasitic Hymenoptera of which 17 (most as principal author ) in international peer -review journals with impact
factor (J Na t Hist , Zookeys , Zootaxa etc.), and one on the “yellow” CNCSIS category ( Invertebrate taxonomy ).
Role : testing artificial media and rearing flies, identifying and rearing Trichopria , curing the innate infections
with antibiotics, maintaining the Trichopria lines .
Maria -Magdalena Dascălu (postdoc): PhD in Biology (Entomology) (2007) . Postdoc in molecular
taxonomy (2010 -2013). Published 1 book, 1 book chapter and 1 6 papers of which 4 in international peer -review
journals with impact factor . Team member in the project s PARASYN and NEVPIT ; masters DNA manipulation
and phylogenetic analysis techniques; knowledge of insect rearing and artificial media. Role : field collecting ,
DNA extraction s, PCR, sequence analyses, maintaining parasitoids in culture .
Viciriuc I. Madalina (PhD student ): MSc in Biology (2016). Published 2 paper s (one with IF) and
presented her results at 3 international and one national conference. Several internships on biological control at
Sophia Agrobiotech (France). Volunteer in the project PAR ASYN and employed by the pro ject NEVPIT.
Undergraduate thesis on optimization o f PCR conditions for DNA barcoding in parasitoid wasps of synanthropic
flies; MSc thesis on the molecular phylogeny of Spalangia ; PhD on integrated taxonomy of parasitoid wasps .
Masters very well techniques of DNA manipulation. Role : DNA extractions, PCR, sequence analyses, assisting
LF for cloning and NGS experiments.
The supervisor of the PhD student is M.D. Mitroiu, member in this grant proposal. The data obtained during
this project ( correlations between parasitoid lineages and Wolbachia infections) will be integrated into her PhD
thesis.
Technician (open position) . We envisage of involving two of our current BSc or MSc students (for rearing
the Pteromalidae and Diapriidae pa rasitoids respectively).

Partner 1 (ICB Iași)
Senior Researcher II Dr Vochița Gabriela (Person in charge from Partner 1) : PhD in biology (2006).
Leader of national and international cooperation projects (JINR – Dubna , since 2011) and member in 24 national
scientif ic project; 1 book and 1 book chapter; 25 papers published in journals with impact factor and 69 in
Romanian BDI journals; participation of international (40) and national (49) conference/congresses . Expertis e in
in vitro cell cultures techniques (has publ ished 12 ISI and 45 BDI articles that include in vitro cell
technologies , ex. Mihai et al. 2014 ), biochemistry tests, animal and plant cytogenetics (karyotype, idiogram,
physical and chemical mutagenesis (chromosomal aberrations, c omet assay, micronuclei t est, Sister Chromatid
Exchanges – SCE technique, fluorescence in situ hybridization – FISH), molecular biology (PCR,
electrophoresis). Role : ensur ing the suitable scientific and financial progress of the project; good collaboration
with Coordinator institu tion by provid ing the scientific and financial reports ; assures the timely acquisition of the
new cell line and materials /reagents for conducting of the planned experiments ; initiation of the new cell line
culture and design of in vitro experiments ; qPCR f or Wolbachia titer .

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PN-III-CERC -CO-PED -2-2019 Senior Researcher III Dr Gherghel Daniela (researcher) : PhD in biology (2003). Romanian responsible
of international cooperation projects (3 projects: JINR – Dubna, since 2015), national project responsible (2
projects) , and member in 28 national scientific projects; 2 book chapters (co -author); 15 papers published in ISI
journals and 47 in Romanian BDI journals, 95 citations in ISI quoted journals (28 with impact factor); patents
for invention (1) . Expertise in cellular and molecular biology; in vitro cell cultures of healthy and neoplastic cells
(9 ISI and 15 BDI articles with studies on in vitro cell line reactivity , ex. Rață et al. 2019 ); elucidation of the
mechanisms of action of various xenobiotics; biochemistry; in vivo screening on hea lthy animals or bearing of
different types of tumou rs; animal physiology; therapeutic strategies. Role: the in vitro experiments with the new
cell line in order to assure its proper reactivity (cell viability, proliferation, etc.) ; propaga tion and sub cult uring
of the cell lines with Wolbachia .

B.2.3 Method of project implementation
The content of this section corresponds to the third evaluation criterion (method of project implementation).

The exact species candidates will be chosen during the initia l phase of the project. Several species of
Spalangia are taken into consideration due to their high par asitism rates and their presence in the Romanian
fauna ( S. nigroaenea Curtis, S. endius Walker, S. cameroni Perkins, S. nigra Latreille) plus Trichopria (T.
sociabilis Masner and possibly other species).
1. Testing and continuous improvement of several variants of artificial diets for rearing Musca and
Stomoxys . We will avoid growing a substitution host for our parasitoids and will try to grow Musca domest ica
(curly wing mutants for ease of manipulation) and Stomoxys calcitrans . This is because w e want to avoid an
adaptation of the wasps to the substitution hosts that might lower the parasitism rates in field settings. We will
use simple artificial media de rived from the protocol of Hogsette (1992) as it is suitable for both of these flies.
We alr eady have experience in rearing insects in captivity (tropical cockroaches, phasmids, house crickets,
bruchid beetles etc.) hence rearing flies will not be a proble m. This artificial medium will contain alfalfa meal,
wheat bran, corn meal, a source of prot ein, brewers’ yeast, brewers dried grains in different proportions. We will
experiment by changing the source of protein (soy meal, powdered milk, meat + bone meal) and by adding
antifungal agents (e.g. methylparaben). This medium is suitable for growing o f Musca domestica . By adding
pelleted peanut hulls in 1:1 volumes, this diet also becomes suitable for Stomoxys calcitrans . We will experiment
to find a replacement for the peanut hulls ( Activity n o 1). Contributing : LF, MDM, OAP, Technician.
2. Obtaining Spalangia and Trichopria parasitoids from fly pupae collected from animal farms and
rearing them on fly pupae in the laboratory.
2.1. Field collections using senti nel fly pupae. Based on the presence data from the PARASYN project we
will collect the model species in the first stage of this project (s ummer of 20 20). We will use freeze -killed or
alive sentinel pupae to collect Spalangia and Trichopria parasitoids from sheds, stables, and manure mounds in
the countryside of NE Romania ( Activity n o 2). Contrib uting : LF, MDM, MMD.
2.2. Rearing parasitoids in the laboratory on fly pupae. Parasitoids will be reared as described by Postu,
Popovici & Mitroiu (2013). The colon ies will be maintained at room temperature in dark well ventilated cages
(excess light cause s lower parasitism percentages). Host fly pupae with adult parasitoids inside will be kept in
vials covered with cotton plugs. A few drops of water are added in eac h vial in order to avoid dryness of pupae
and parasitoids. 200 host pupae (ether freeze -killed or alive) no older than 4 -5 hours (they need to be soft in
order to be accepted by parasitoids) will be placed in contact with 25 -30 parasitized pupae (fewer for Trichopria ,
as they are gregarious parasitoids) in one rearing vial. After emerging, adult parasitoids will mate and then
females will lay eggs in the new pupae. Emerging parasitoids will be removed with a forceps and moved to other
rearing vials. ( Activi ty no 3). Contributing : LF, MDM, OAP, Technician.
3. Testing the candidate species for the presence of endosymbionts and cure the innate infections.
3.1. Testing the infection status of candidate species with TA cloning and Sanger sequencing or
metagenomi cs. Spalangia are known to harbor Wolbachia (Floate et al. 2008). Since nothing is know n about
other endosymbionts that might interfere with our experiments the innate symbiotic microbiota of the candidate
species will be screened using either a cloning an d sequencing approach (low number of species/specimens), or
if a large number of specie s will be screened – using next generation sequencing (NGS) (for large numbers of
amplicons that are multiplexed, NGS can be similarly priced as Sanger sequencing, e.g. about 1500 Euro

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PN-III-CERC -CO-PED -2-2019 estimated per 96 samples). DNA will be extracted as described by us (Cr uaud et al. 2019) as it is suitable for
both Sanger and NGS. For the cloning approach we will PCR amplify the bacterial 16S using universal primers
27F (5′ -AGA GTT TGA T CM TGG CTC AG -3′) and 1513R (5′ -ACG GYT ACC TTG TTA CGA CTT -3′),
clone the product into E. coli competent cells (e.g. TOP10, JM109) by TA cloning (we have positive experience
with the pGEM -T Easy plasmid vector, Promega), check for the presence of the inse rt by colony PCR, inserts of
the correct size will be amplified with nested universal b acterial primers 341f (5′ -CCT ACG GGA GGC AGC
AG-3′) and 907r (5′ -CCG TCA ATT CMT TTG AGT TT -3′) and sequenced at a sequencing facility (e.g.
Macrogen). For the NGS appr oach we will PCR amplify the 16S V2 and V3 regions of the microbial genome
with primers with overhang adapters, attach dual -indices (multiplex identifier tag sequence) and Illumina
sequencing adapters (e.g. with NEBNext Multiplex Oligos for Illumina or Nex tera Index Kit), quantify the
libraries, pool libraries at equimolar concentrations for sequencing and sequence using paired 300‐bp reads at a
sequencing facility (e.g. Macrogen). Reads will be sorted by host species based on the index sequence, and after
trimming the barcodes and primers (e.g. with prinseq ) each dataset will be compared to the Ribosomal Database
Project (RDP) (Activity n o 4). Contributing : LF, MDM, MMD, MV.
Hence, d epending the outcome of Activity 2 .1. (small versus large number of wasps) we will have a cloning
plus Sanger approach or an NGS approach. If the NGS approach wi ll be chosen, the PhD student and one
researcher will undergo a training on library preparation at CBGP (Montpellier, France) with whom we have
a longtime collaboration (Al khatib et al. 2014 and 2016 , Cruaud et al. 2019) .
3.2. Curing the candidate lines. We will try two antibiotics ( tetracycline or rifampicin) at different
concentrations using established methods (e.g. Zchori -Fein et al . 2000; Giorgini, 2001). 0.1 to 20 mg/ml of
tetracycline or rifampicin added to a syrup of honey with water will be fed to adult females for a few days before
offering them fly pupae to oviposit. The effect of the cure will be tested on the progeny by PCR with specific
primers ( Cardinium , Wolbachia , and Rickettsia ) depending on the infection status information gained from
activity no 4 (Activity n o 5). Contributing : LF, MDM, OAP.
3.3. The above results will be presented at a conference and published ( Activity no 6 ). Contributing : LF,
MDM, OAP, MMD, MV, GV, DG.
4. We will use several sources of Wolbachia for our experiment. Pro bably the most promising one is the
thelytokous Muscidifurax uniraptor (Pteromalidae) but we will also try alternative sources such as Diplolepis
wasps (Cynipidae). Methods for transferring Wolbachia endosymbionts from one host to a nother are relatively
well established (Grenier et al. 1998; van Meer & Stouthamer 1999; Watanabe, Kageyama & Miura 2013).
4.1. Obtaining Drosophila cell cultures infected with the desired Wolbachia strain. This activity will be
accomplished by Partner 1 who h as the necessary facilit ies for anima cell culture. After surface sterilization of
the donor insect, ovaries will be extracted by microdissection (as described in Fusu, 2008 or Popovici & Johnson
2012) on a depression slide in a drop of sterile culture med ium. The tissue will be homogenized and transferred
to a cell culture. We will use a well -researched insect cell line (Schneider's Drosophila Line 2). The cell culture
will be grown in Schneider's Drosophila Medium supplemented with 10% heat inactivated FB S, in an incubator
at 23 °C without CO2. Cells will be subcultivated several times (1:10 subcultivation ratio) and tested for
Wolbachia infection with specific primers. The cells will be cultivated throughout the project and used as a
source of Wolbachia (a cell culture permits to adjust the density of the infectious agent, which is not possible if
cells from an infected insect are used directly); stocks cultures will be cryopreserved ( Activity n o 7).
Contributing : LF, GV, DG .
4.2. Transinfections with Wolbachia , and screening with qPCR of the established isofemale lines.
Parasitoid pupae or prepupae will be removed from the puparium and transinfected with Wolbachia using a
microinjection with cell suspension from the infected Schnei der 2 cell line. Surviving resulting females will be
mated and isofemale lines established (probably they will not become thelytokous from the first generation).
After reproduction, the progeny of each female will be screened for infection and the Wolbachi a titer determined
using quantitative PCR with 16S Wolbachia primers and a single -stranded DNA probe with a reporter dye and a
quencher. Results will be analyzed in R. We will also perform diagnostic PCR using specific Wolbachia primers
(e.g. for the wsp gene) to confirm that the same Wolbachia strain is present in the donor and the acceptor species
by sequencing the fragment. After confirmation, mere observation of a band of expected size on a gel will be
enough to confirm infection ( Activity n o 8). Contri buting : LF, GV, DG .

9
PN-III-CERC -CO-PED -2-2019 4.4. Selection for a female -biased sex ratio and high Wolbachia titer. We will maintain the lines in culture
while continually selecting for a female -biased sex ratio and high Wolbachia titer, until hopefully at least one
thelytokous line will be obtained ( Activity n o 9). Contributing : LF, MDM, OAP, M MD, MV, Technician.
4.5. Molecular characterization of the newly obtained lines of parasitoid wasps . DNA will be extracted
as described by us (Cruaud et al. 2019) as it leaves the voucher specimens intact. We will characterize the lines
using the CO 1 sequence following the standards established in the project PARASYN for species identification
(primer pair COI pF2/ COI 2437d) plus the ITS2 sequence for a more accurate identification of the strain (primer
pair ITS-F/ ITS -R2): ITS is known to be very variable at least in Spalangia and probably also in Trichopria .
These two sequences plus the sequence of the transfected Wolbachia endosymb iont will provide a unique
“signature” of each strain that can be used to recognize it in field settings and differentiate it from conspecific
wild individuals ( Activity no 10). Contributing : LF, MDM, MV.
4.5. If succesfull , we will fill a patent application for a Wolbachia induced thelytokous line (Activity no 11).
Contributing : LF, MDM, OAP, MMD, GV, DG.

Project schedule with intended start and finish dates of activities
Objec –
tive Acti-
vity Months
2020 2021 2022
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Obj. 1

2
A1
Obj. 2 A2
A3
Obj. 3 A4
A5
A6
Obj. 4 A7
A8
A9
A10
A11

Field work
Insect s rearing and testing
Insect cell lines culture
Molecular chracterisation
Dissemination and intell ectual proper ty rights protection

Detailed work plan. Objectives, associated activities and deliverables
Objectives (O) Activities (A) Deliverable / type* / due date
1. 1. Rearing fly larvae on
artificial diet. A1. Testing and continuous
improvement of several variant s of
artificial diets for rearing Musca and
Stomoxys. At least one affordable and
potentially of commercial usage
recipe for rearing flies / R /
Month 8;
Two recipes / R / Month 20
2. To establish and
propagate one to several
local lines of parasitoid
wasps of synanthropic
flies. A2. Additional field collections using
sentinel fly pupae.
Collecting in at least 5 localities
/ R / Month 5
A3. Rearing parasitoids in the
laboratory on fly pupae. At least two local line of
Spalangia and two of Trichopria
will be established / R / Month s
8, 24.

10
PN-III-CERC -CO-PED -2-2019 3. To test the candidate
species for the presence
of endosymbionts and
cure the innate infection. A4. Testing the infection status of
candidate species with TA cloning and
Sanger sequencing or metagenomics.
Testing at least 4 lines / R /
Month 8 .
A5. Curing the candidate lines with
antibiotics. At least 2 lines cured / R / Month
13.
A6. Presentation of the results at an
international conference and
submission of a scientific paper to a
major peer review journal.
Presentation and publication on
the innate infections and curing
effect of at least two local line of
Spalangia and two of Trichopria
/ S / Month s 16 (submission) and
18 (presentation) .
4. To obtain improved
lines of thelytokous
females artificially
infect ed with Wolbachia
and characterized using
genetic markers. A7. Obtaining Drosophila cell cultures
infected with the desired Wolbachia
strain. A Drosophyla cell culture
adapted to our protocols and
infected with four Wolbachia
strains (four subcultures) / R /
Month 10.
A8. Transinfections with Wolbachia ,
and screening with qPCR of the
established isofemale lines. At least two artificially infected
and genetically characterized
isofemale line s / R / Month 1 1.
A9. Selection for a female -biased sex
ratio an d high Wolbachia titer. Two selected lines / R / Month
21.
A10. Molecular characterization of the
newly obtained lines of parasitoid
wasps. Characterization on two markers
for the wasp and one marker for
the endosymbiont / R / Month
23.
A11. Filing patent applications. Potentially at least one
patentable thelytokous line / R &
P / Month 24.
* P – patent application; R – report; S – scientific publication.

Dissemination of results and intellectual property rights
Because the lines of thelytokous paras itic wasps are a novel product that combines an insect and a
bacterium, and not simply wild -caught specimens reproduced in mass in laboratory or a commercial facility,
they can be patented. Inventions which concern plants or animals are patentable provided that the application of
the invention is not technically confined to a single plant or animal variety (Directive 98/44/EC, Art. 29). Hence
our f inal goal is to obtain patents for these lines, if their establishment will succeed. We intend to submit at lea st
one patent application at O.S.I.M. The scientific results of the analysis of innate infection with endosymbionts
and of the outcomes of curing and artificial infections with Wolbachia have the potential of being published in
leading journals (at least o ne accepted or submitted paper e.g. in Journal of Invertebrate Pathology , Zoological
Journal of the Linnean Society, Bulletin of Entomological Re search ). We will present the results at the CE 2021:
15th International Conference on Entomology , October 29 -30, 2021 in Paris, France

Research infrastructure available for the project (including the link to www.erris.gov.ro ) and its development
during the project
The project will benefit from the infrastructure of the CERNE SIM center as two members of the team of
the present proposal (leading researcher LF and OP) are members of the scientific team of this research
infrastructure center (Integrated Center of Environmental Science Studies in the North East Region;
http://erris.gov.ro/cernesim.uaic.ro ; Service name: Exploration of resources for bioremediation and bio -control ).
The center has rearing facilities (climate chamber EDC01E, Snijders Labs, Netherland), specimen imag ing
facilities (Leica DFC 500 digital camera attached to a Leica M205 A motorised stereomicroscope), facilities for
specimen identification, samples sorting and manipulations (Leica S6) and a laboratory dedicated to insect

11
PN-III-CERC -CO-PED -2-2019 molecular biology and integrative taxonomy. It will also b enefit from the infrastructure generated by the
PARASYN – PNII-RU-TE-2012-3-0057 grant ( Spalangia vouchers collection, Trichopria collection,
thermocycler , centrifuge, electrophoresis and gel imaging equipment, reagents and labware for DNA work,
cages for rearing insects etc.) and the NEVPIT – PN-III-P4-ID-PCE-2016 -0233 grant (rearing cages, Leica
DFC450 high resolution camera , Leica LED5000 HDI light source .
Available infrastructure and resources : Insect culture. (1) Climate chambe r EDC01E; (2) Climatic hood
810 for parasitic wasps. Main equipment and facilities for cell culture. (1) Laminar flow hood Sterile Helios;
(2) Mini -incubator Labnet. Main equipment and facilities for genetic analyses. (1) Labcycler thermocycler w
gradient; (2) LightCycler 2.0, Roche Life Science for qPCR; (3) E lectrophoresis supply Consort EV231,
horizontal gel systems MultiSUB Choice and Scie -Plas; (4) Transilluminator Maestrogen 302/365nm with
imaging equipment; (5) Freezers and GFL deep freezer; (6) Tabl etop centrifuge Eppendorf 5424; (7) Julabo
water bath; ( 8) Raypa autoclave; (9) Sets of Gilson variable volume pipettes etc. Species identification,
specimen preparation, dissections and imaging. (1) Large collection of parasitoids of synanthropic flies fr om
the project PARASYN; (2) Critical point dryer EMS 850 ; (3) Automated research stereomicroscope Leica M205
A with Leica DFC500 and DFC450 high resolution camera s; (4) Several Leica and Olympus SZX9
stereomicrosopes; (5) EMS 550X Sputter Coater; (6) scann ing electronic microscope Vega 3SB, Tescan ; (7)
Leica LED5000 HDI light source .
Requested equipment . The existing infrastructure allows us to start the project without delays, but it will be
necessary to acquire some additional equipment to optimize the w orkflow: a microinjector (e.g. CellTram ® Oil;
7770 RON) for the transfection experiments .

Structure of the research team and justification of salary expenses
Team members Institu –
tion Activities Person
months/
member Salary*
Lucian Fusu (principal inves tigator) UAIC A1-A11 12 52800
Mircea D. Mitroiu (researcher) UAIC A1-A6, A9, A11 12 38400
Ovidiu A. Popovici (researcher) UAIC A1, A3, A5, A6, A9, A11 12 38400
Maria -Magdalena Dascălu (postdoc) UAIC A2, A4, A6, A9, A10 -11 24 96000
Madalina Viciriuc ( PhD student ) UAIC A4, A6, A9, A10 21 67200
Technician UAIC A1, A3, A9 6 28800
Gabriela Vochița (researcher, in charge for P1) ICB-Iași
(P1) A6, A7, A8, A11 10 35562
Daniela Gherghel (researcher) ICB-Iași
(P1) A6, A7, A8 , A11 10 32638
* Gross earnings plus employer payroll taxes

Risks associated with the project implementation and ways of treating them
Activity Potential risks
/ likelihood Impact on project
execution by objective Risk management
A1
Failure in growing
synanthropic flies in the
laboratory / Low risk. Low to inexistent. There are numerous alternatives of
artificial diets for fly larvae to find
one that will fit our needs. Besides,
fly larvae can b e purchased
commercially.
A2
A3 Failure in establishing a
stable laboratory line of
parasitic wasps / Low risk. High, the project will fail
from the start. All following
objectives will fail. Trichopria reproduces well under
laboratory conditions as was a lready
demonstrated by us, so there is a
virtually zero risk for this species.
By testing several species of
Spalangia we can choose at least
one.

12
PN-III-CERC -CO-PED -2-2019 A5 Failure of antibiotic
treatment / Low risk. High impact for the
development of an improved
thelytokous li ne (Obj.4), but
no impact on the production
of locally adapted lines
(Obj.2). We envisage to use at least two
antibiotics, of which at least o ne
should work.
A7
A8 Failure of obtaining lines of
parasitic wasp infected with
a horizontally transferred
Wolba chia / Low to medium
risk. High impact for the
development of an improved
thelytokous line (Obj.4), but
no impact on the production
of locally adapted lines
(Obj.2). We plan to use several potential
sources (donor species) of thelytoky –
inducing Wolbachia , to minimize
and even eliminate the risk of failure
at this stage.
A9
A10 Failure in obtaining at least
one stable thelytokous line /
High risk. High impact for the
development of an improved
thelytokous line (Obj.4), but
no impact on the production
of loc ally adapted lines
(Obj.2). If the last step in the project fails,
only one of the two main objectives
will not be attained (improved
thely tokous line), but we will still
obtain at least one locally adapted
line of parasitic wasps to control
synanthropic f lies.

Project Budget (not detailed by year, as suggested by the provided template) :

Logistics for CO include 7 770 in equipment ( a microinjector ) and 45 000 in kits (20 000) , laboratory plastics
(10 000) , sequenc ing costs (15 000) .
Logistics for P1 include 26 000 in growing media and reagents (18 000), cell lines acquisition (8 000).

Travel for CO includes participation of two persons at a conference ( 6 000), a training at CBGP (France) (6
000), local travel for sampling ( 3 000).
Travel for P1 includes participation of two persons at a conference (6 000)

1 Subcontracting – no more than 5% of the project’s public budget
2 For institutions under the state aid scheme, costs f or travel will be made from their own contribution
3 Max. 25% of direct costs minus subcontracting and equipment costs. Allocated budget / costs (Lei)
Personal
costs Logistics1 Travel2 Indirect costs3 Total
Coordinator
(CO) Public budget 321600 52770 15000 95400 484770
Partener 1
(P1) Public budget 68200 26000 6000 15030 115230
Own
contribution (if
applicable)
Total 389800 78770 21000 110430 600000

13
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15
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