The use of nest boxes in determination of biting midges involved in transmission of Haemoproteus parasites

Culicoides biting midges (Diptera, Ceratopogonidae) are known to be vectors of avian Haemoproteus parasites. These parasites cause disease and pathology in birds. The diversity of biting midges in Europe is great, but only four Culicoides species are known to be vectors of avian Haemoproteus parasites. In general, our knowledge about the role of the particular Culicoides species in transmission of Haemoproteus parasites remains insufficient. Information gaps hinder a better understanding of parasite biology and the epizootiology of parasite-caused diseases. The aim of this study was to determine new ornithophilic Culicoides species potentially involved in local transmission of Haemoproteus parasites. To do this we collected biting midges in bird nest boxes, identified their species and prevalence of haemoproteids in insects as well as in juvenile birds during breeding season. Biting midges were collected from bird nest boxes, identified and parous females were tested individually The blood of juvenile birds was sampled determine the study We have used both microscopy and PCR-based methods.


Conclusions
Our results provided information about the distribution of biting midge species and determined new ornithophilic Culicoides species at the study site. This study contributes to epizootiology of avian Haemoproteus infections by specifying Culicoides species that likely are responsible for the transmission of haemoproteids in Europe. Used method is suitable for better understanding vector ecology and evaluating the role of different blood sucking insects in transmission of haemoproteids in different wild ecosystems.

Background
Blood-sucking biting midges (Ceratopogonidae: Culicoides) play an important role in various wildlife processes, they are transmitters of viruses, bacteria, parasitic protozoa and nematodes [1][2][3]. They are vectors of the Haemoproteus (Haemosporida) parasites, which can cause diseases and even lethal pathology in non-adapted birds [1][2][3][4][5]. At present there are 1368 species of biting midges known in the world [6], but only 12 of them have been proved to support complete sporogony of avian Haemoproteus parasites [7,8], though about 150 species of Haemoproteus have been described [9]. Culicoides impunctatus is one of the most abundant species in North Europe [11,12], therefor the most exhaustive experimental studies on sporogony of haemoproteids have been done with wild C. 4 impunctatus biting midges [2,10]. Other experimental studies were performed with Culicoides nubeculosus, which is the only Palearctic Culicoides species cultivated in laboratories [7,13]. Recently, Bernotienė et al., [8] [3]. However, most of these biting midge species except C. sphangnumensis are distributed in North America and cannot be found in Europe.
Several recent studies have reported other Culicoides species as possible Haemoproteus vectors, but their analyses were based only on molecular determination of the parasite DNA obtained from wild-caught insects [14][15][16][17][18]. PCRbased studies are helpful in determination of ornithophilic biting midges [19] and this information is essential in vector research. Presence of parasite's DNA indicates that the insect might be possible vector, but these data alone are insufficient to prove the transmission of haemoproteids, because PCR-based diagnostics cannot distinguish between invasive for vertebrate hosts (sporozoites) and non-invasive sporogonic stages [20]. Our knowledge about the composition of Culicoides species, which may be involved in transmission of Haemoproteus parasites in wild is insufficient and it interferes for better understanding patterns of epzootiology of vector-borne diseases in wildlife [2,3,21]. Therefore, the aim of this study was to identify ornithophilic biting midges obtained from bird nest boxes and to determine which species of Culicoides could potentially participate in the transmission of  [2]. This short and vulnerable for the host period was used in our study. First, we have collected midges from nest boxes as described by Tomas et al. [22]. Biting midges of these species were intentionally looking for the bird blood, so were ornithophilic and could potentially play a role in transmission network of Haemoproteus parasites. Second, to find out if caught biting midges contained DNA of Haemoproteus spp., we applied PCR-based analysis for each of collected insect. Third, to determine, which haemosporidian parasites are transmitted at study areas we checked juvenile birds

Study site
This study was carried out at the Neris Regional park (NRP), Lithuania (54°50'N

Collection of biting midges and microscopic examination
Blood-sucking insects were collected according to methodology described by Tomas 6 et al. [22]. Nest boxes were attached to the tree up to 2 meters high ( Fig. 1 a). Petri dishes moisten with baby oil were temporarily fixed upside down using double sided sticky tape on inside roofs of nest boxes (Fig. 1 c). Insects flying inside the nest boxes stuck to the Petri dishes, thus could be collected (Fig. d). Seventy three nest boxes with nesting birds and hatched nestlings were investigated: eleven in NRP and sixty-two in BSR (14, 28 and 20 in 2012, 2018 and 2019, respectively).
Petri dishes were left overnight and were removed next day, because Culicoides biting midges are active at dusk [12]. Petri dishes were replaced several times per week from the end of May to the end of July. Petri dishes with sticking insects were taken to the laboratory, biting midges were anesthetized with 96% ethanol, identified according to the morphological features [23][24][25]. The heads of biting midges were removed to prepare permanent preparations mounted in Euparal for the further identification of Culicoides species.
Parous females were detected according to the presence of the readily visible burgundy pigment in the subcutaneous cells of the abdomen, indicating a digested blood meal prior to capture [26]. These females were dissected for preparation of salivary glands as described by [2,27]. Invasive stage of haemoproteids (sporozoites) can be found in salivary glands of infected biting midges [2]. Briefly, insects were placed in a small drop of 0.9 % normal saline. Head and thorax were separated. Extracted salivary glands were grinded using a needle and mixed with a tiny drop of saline. Preparations were dried in the air, fixed with absolute methanol, and stained with 4% Giemsa stain. Remnants of insects were placed for PCR-based confirmation of the insect species and the detection of parasite's DNA in insect using primers for haemosporidian parasites (as described below) [28][29][30]. To eliminate contamination of samples, we used a new dissecting needle for each 7 dissected biting midge. All material was studied under the binocular stereoscopic microscope Olympus SZX10 and Olympus BX43 microscope (Tokyo, Japan).

Collection of juvenile bird blood and microscopic examination
In order to detect haemoproteids which are transmitted in the study area, the juvenile birds of the species which usually nesting in nest boxes (Parus major, Poecile palustris , Cyenistes caeruleus, Ficedula hypoleuca) were captured by mist nets during fieldwork. About 50 μl of blood was taken in heparinized microcapillaries by puncturing the brachial vein. A small drop was used for preparation of two blood smears from each individual. Residual blood was stored in SET-buffer for molecular analysis [31]. The smears were air-dried, fixed in absolute methanol and stained with Giemsa stain solution, as described by [32]. Approximately 100-150 fields were examined at low magnification (400×), and then at least 100 fields were studied at high magnification (1000×). The intensity of parasitemia was estimated as a percentage by actual counting of the number of parasites per 1000 erythrocytes or per 10,000 erythrocytes if infections were light (< 0.1%), as recommended by Godfrey et al. [33]. We used an Olympus BX43 light microscope (Tokyo, Japan) to analyze the blood slides. Haemoproteus parasites were identified according to Valkiūnas [2]. All birds were released in the same area as captured after blood sampling.

Polymerase chain reaction and sequencing
Total DNA was extracted from each individual biting midge and from the bird blood using ammonium acetate DNA extraction method [34].
For detection of haemoproteids the segment of parasite mitochondrial cytochrome b (cyt b) gene fragment was amplified using nested PCR protocol [29,30]. The initial 8 primers HaemNFI and HaemNR3 and nested primers HaemF and HaemR2 were used.
To confirm identification of Culicoides species we used insect specific primers LCO149 and HCO2198 to amplify a fragment of cytochrome c oxidase subunit 1 (cox1) of mitochondrial DNA [28]. Morphological identification was consistent with PCR-based identification of biting midges.
Fragments of DNA of all PCR positive samples were visualized on 2% agarose gel using MidoriGreen dye (NIPPON Genetics Europe, Germany). All positive samples were sequenced using corresponding primers and sequences were edited and aligned using BioEdit program [35].  Table 1). The abundance of biting midges was low in 2019 in spite of the fact that the timing and the same sampling method was used (Table 1). This year the most abundant species (30,4%) was C. kibunensis.

Discussion
It is known that sporogony of different Haemoproteus species can take place in four European Culicoides species: C. impunctatus, C. nubeculosus, C. kibunensis and C.

sphagnumensis. Culicoides impunctatus is one of the most abundant species of
Culicoides in North Europe as well as in our study site [12,38]. This species is excellent experimental vector and likely is natural vector of 12 species of Haemoproteus parasites [10]. Biting midges of this Culicoides species are extremely abundant in June in some localities and this allows using them in experimental research [2,10]. Culicoides impunctatus used to be considered as mammalophilic species [39], but cases of ornithophilic behavior of these biting midges have been documented [10]. Our study confirms ornithophilic C. impunctatus behavior as this insect was found visiting nest boxes of breeding birds in our study.

Culicoides kibunensis was detected as a vector of Haemoproteus pallidus in
Lithuania, because two wild caught individuals of this species were detected to harbor DNA as well as sporozoites of H. pallidus (lineage hPCF1) [8]. Culicoides nubeculosus is the only Palearctic Culicoides species cultivated in laboratory, that is why, experimental studies on sporogony of several Haemoproteus spp. in these biting midges were performed in recent years [7,13]. Some studies followed sporogony of haemoproteids till the sporozoite stage in C. sphagnumensis [3].The diversity of Culicoides in the Europe is high -more than 100 species are known [25] and there is no information about other Culicoides species which would be known as Birds, at nesting time are easy targets for blood sucking insects [2], so the collection of insects in bird nest boxes can help both to determine ornithophilic insect species and to identify infected biting midges. The host range of biting midges is difficult to determine and it remains insufficiently investigated. Additional data providing information about host preference of Culicoides biting midges are important for epizootiology studies. Five out of 11 Culicoides species, collected in nest boxes, have been already known to take blood meals on birds: Culicoides kibunensis, C. pictipennis, C. segnis, C. impunctatus and C. festivipennis [10, 15-17, 21, 40], but only sporadic cases of ornithophily have been reported for biting midges belonging to other species [40]. Culicoides reconditus, C. subfascipennis, C. pallidicornis, C. punctatus and C. obsoletus were also collected in nest boxes thus they likely naturally were looking for bird blood in the wild.
Culicoides obsoletus and C. punctatus are among the most abundant biting midges in North Europe [41,42], thus they should be considered for experimental research as potential vector candidates for Haemoproteus transmission. Culicoides kibunensis, C. segnis and C. pictipennis being the dominant species attacking birds, as determined in this study, were not known to be abundant at study site. It was documented that C. impunctatus was the most dominant species in the Curonian spit and formed 82.1 -99.7 % of all Culicoides [12, 43,44] and this species is still the dominant in some localities of the Curonian spit in June. Culicoides species being the dominant in nest boxes according to our data were even not detected using other collection methods (light, netting, collection from humans) on the Curonian spit during earlier investigations [12,43]. The method applied for insect collection may have crucial impact on species composition and abundance of collected insects, this method to collect biting midges from nest boxes may be of great importance with the target to find ornithophilic species and potential vectors of avian haemoproteids.
PCR-based testing of wild-caught insects for the presence of Haemoproteus DNA can also be helpful in detecting potential vectors of avian haemoproteids, but this method alone is insufficient to demonstrate that the insect is a vector of the parasite [19]. Experimental studies indicate that avian malaria parasites can persist even in resistant blood sucking insects for several weeks after initial blood meals due to the survival of ookinetes. These parasites can be gained only during infected blood meals on birds, so the presence of parasite's DNA proves only that biting 13 midge have taken blood meal from the bird before [20]. According to PCR-based testing, nine Culicoides species are known to harbor Haemoproteus parasite DNA in Europe. These are Culicoides alazanicus [15], Culicoides circumscriptus, C.
segnis [16], C. scoticus [8,17], C. punctatus and C. obsoletus [8].We have detected avian haemosporidian parasites in biting midges belonging to 5 Culicoides species and have added C. reconditus to this list ( Culicoides punctatus and C. pictipennis females were found in nest boxes and were infected with H. minutus (hTURDUS2 and hTUPHI01, respectively) ( Table 1). These parasites are widespread in common blackbirds Turdus merula in Europe and in our study site [46]. Haemoproteus minutus cause mortality in captive parrots in Europe by causing lethal disease on the stage of megalomeronts [45,47]. It was shown that H. minutus (hTURDUS2) can be transmitted by C. impunctatus [27] and laboratory cultivated C. nubeculosus biting midges [7]. Known data indicate broad susceptibility of the C. impunctatus and C. nubeculosus biting midges to many Haemoproteus parasites [7,10] and in general shows low vector specificity of the

Conclusion
The application of the new collection method revealed the species composition of 15 ornithophilic Culicoides biting midges. Results obtained from the blood of juvenile birds have shown that transmission of H. majoris (hCWT4, hPARUS1, hPHSIB1, hWW2) and Haemoproteus sp. (hPARUS10) parasites take place at our study site.
Culicoides segnis, C. pictipennis, and C. kibunensis being the dominant ornithophilic species and found to be infected with Haemoproteus parasites should be considered as possible vectors of these parasites.
The data on the possible Haemoproteus vectors can help to initiate detailed experimental studies of sporogony of various Haemoproteus spp. parasites with the most abundant ornithophilic biting midge species. Obtained information will be valuable for the knowledge on the vectors of haemosporidian parasites in Europe.

Ethics approval
Experimental procedures of this study were approved by the International Research Cooperation Agreement between the Zoological Institute of the Russian Academy of Sciences and the Nature Research Centre (1-12-2015-30-11-2020). All efforts were made to minimize handling time and potential suffering of animals.

Consent of publication
Not applicable.

Availability of data and materials
The data that support findings of this study are included within the article.

Competing interests
The authors declare that they have no competing interests.