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Article

What Can Haemosporidian Lineages Found in Culicoides Biting Midges Tell Us about Their Feeding Preferences?

by
Carolina Romeiro Fernandes Chagas
*,
Carolina Hernández-Lara
,
Mélanie Duc
,
Kristina Valavičiūtė-Pocienė
and
Rasa Bernotienė
Nature Research Centre, Akademijos 2, 08412 Vilnius, Lithuania
*
Author to whom correspondence should be addressed.
Diversity 2022, 14(11), 957; https://0-doi-org.brum.beds.ac.uk/10.3390/d14110957
Submission received: 5 October 2022 / Revised: 3 November 2022 / Accepted: 3 November 2022 / Published: 8 November 2022
(This article belongs to the Special Issue Diversity of Wildlife Pathogens)
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Abstract

:
Haemoproteus (Parahaemoproteus) parasites are transmitted by Culicoides biting midges. However, the natural vectors of only six of the almost 180 recognized Haemoproteus species have been identified. The aim of this study was to investigate wild biting midges naturally infected with Haemoproteus and to understand the interaction network between Culicoides and Haemoproteus in Europe. Culicoides were collected with UV light traps from different sites in Lithuania. Parous females were morphologically identified based on their wings and heads. PCR-based methods were used to detect the Haemoproteus DNA, and salivary gland preparations were analyzed for the presence of sporozoites. Of the 580 Culicoides analyzed, 5.9% were positive for Haemoproteus DNA, and sporozoites were found in two of 11 sampled biting midge species: Culicoides kibunensis and Culicoides segnis. The interaction network revealed that C. kibunensis and C. segnis are frequently associated with several Haemoproteus lineages. On the other hand, some Haemoproteus lineages were found to interact with only one Culicoides species. This was the first report of C. segnis being a competent vector for H. minutus TURDUS2, H. asymmetricus TUPHI01, H. majoris PHSIB1, and H. fringillae CCF3; and of C. kibunensis being a competent vector for H. belopolskyi HIICT1. Culicoides segnis and C. kibunensis are both important vectors of Haemoproteus parasites.

Graphical Abstract

1. Introduction

Haemoproteus (Parahaemoproteus) parasites are commonly found in birds, sometimes causing serious diseases and even leading to the high mortality of infected individuals [1]. They are transmitted by Culicoides biting midges; however, little is known about their natural vectors. There are almost 180 described species of Haemoproteus parasites [1], and more than 1400 species of Culicoides worldwide [2]; however, the natural vectors of Haemoproteus are known for only a small portion of parasite species [3,4,5,6]. Further, considering only studies combining the investigation of sporozoites in salivary gland preparations and using molecular tools to confirm parasite species and lineage, this number is even smaller [3,4,5,6].
Only 14 species of biting midges have been confirmed to be PCR-positive for Haemoproteus parasite DNA in Europe [3,5,6,7,8,9,10,11,12], which merely indicates that they had fed on infected birds, but not that the insect is a competent vector for the parasite. For this confirmation, it is necessary to prove the presence of sporozoites (the infective stage of haemosporidian parasites) in the insect salivary glands [13], which, in natural infections, was done for only six species of Haemoproteus (H. pallidus cytochrome b lineage PFC1, H. parabelopolskyi SYAT02, H. majoris CCF5, H. tartakovskyi HAWF1, H. minutus TURDUS2, and H. asymmetricus TUPHI01) and three species of Culicoides (C. kibunensis, C. pictipennis, and C. segnis) [3,5,6].
Additionally, many experimental studies have been conducted to investigate Haemoproteus development in Culicoides biting midges [4,14,15,16]. Even though they were able to prove that the parasite can develop in a certain Culicoides species, this does not mean that it is what occurs in nature. If the Culicoides species does not naturally feed upon the infected vertebrate host, it will not acquire the parasite, or transmit it. For example, the majority of these experimental studies have been conducted with C. impunctatus and C. nubeculosus [4], but so far, these two biting midge species have not been proven to be natural vectors of Haemoproteus parasites [3,5,6], probably because they are mammalophilic biting midge species, feeding on birds only opportunistically [17,18,19].
Understanding the transmission of vector-borne diseases is not so straightforward, given that the insect must first be interested in biting an infected bird, and then survive until the parasite completes its development, which means that the infective stages of the parasite are present in the salivary glands of the insect (in the case of haemosporidians), and finally feeds on a susceptible host [20]. However, many other factors are involved in this interaction, such as the feeding preference, reproductive biology, and attack rates of insects, as well as the defensive behavior, age, species, and health of the hosts [20]. It is also necessary to mention that parasites are evolutionarily adapted to evade the host and vector immune systems, replicate in host tissues, and surpass infection and transmission barriers in the insect body, which can also interfere in the transmission rates of vector-borne diseases [21].
This shows how complex host-parasite-vector relationships can be and how important it is to focus on studies targeting these relationships to understand the natural transmission of avian blood parasites. This study aimed to demonstrate the vector competence of Culicoides biting midges naturally infected with Haemoproteus parasites and thereby confirm their role as natural vectors of these avian parasites. Additionally, we also compared the interaction network between Culicoides biting midges and Haemoproteus lineages in Europe to better understand the parasite-vector relationship. For this, we considered all studies published to date in Europe reporting Culicoides biting midges that were PCR-positive for Haemoproteus DNA.

2. Materials and Methods

2.1. Biting Midges Collection

Biting midges were collected between June and September 2021 using UV LED-light traps (BG-Pro All-In-One Biogents AG) in different regions of Lithuania: Verkiai Regional Park (54°45′00″ N, 25°17′00″ E), Vilnius University Botanical Garden (54°44′12.5″ N 25°24′16.4″ E), Puvočiai (54°06′52.2″ N 24°18′17.6″ E), Ventės Ragas (55°20′28.1″ N, 21°11′25.3″ E) and its surroundings (55°23′57.5″ N 21°14′14.8″ E and 55°26′12.0″ N 21°16′04.6″ E) (Figure 1). The study sites were chosen due to their proximity to water bodies (such as lagoons and rivers), as well as their soil humidity, closed woods, and lack of wind. The traps were hung 6–7 h before sunset and removed 4–5 h after sunrise. Biting midges were collected into a small pot containing water with a drop of liquid soap.
Samples were transported to the laboratory right after collection for processing. The fresh material was investigated under a binocular stereo microscope, and female Culicoides biting midges with burgundy pigment (which indicates that at least one gonotrophic cycle occurred, meaning that there is a greater chance that the insect had, at least, one blood meal [22] and thus, was more likely to yield sporozoites of haemosporidian parasites) were dissected for salivary gland preparations.

2.2. Biting Midges Dissection, Identification, and Microscopic Examination of Salivary Gland Preparations

During dissection, each insect was placed in a drop of 0.9% saline solution on a glass slide. The head and wings were removed and transferred to a new glass slide containing a small drop of Euparal, covered with a cover slide, and dried at room temperature for two months or in an incubator at 60 °C for one week. These permanent preparations were then used for morphological identification of dissected Culicoides [23,24,25].
The salivary glands are in the anterior/upper part of the insect’s thorax [13], which was gently crushed using dissecting needles to prepare a small thin smear [26]. To avoid contamination, all dissecting needles were disinfected in fire after each dissection. The salivary gland preparations were air dried, fixed with a drop of absolute methanol, and stained with a 4% Giemsa solution [13,26]. Remnants of dissected biting midges were stored in 96% alcohol for PCR-based analysis.
All salivary gland preparations of insects that were PCR-positive for Haemoproteus parasite DNA were examined using an Olympus BX-43 light microscope equipped with an Olympus DP12 digital camera and the image software Olympus DP-SOFT (Olympus, Tokyo, Japan). The entire smear was examined at high magnification (1000×). Representative preparations of sporozoites (accession nos. 49409NS-49425NS) were deposited at the Nature Research Centre, Vilnius, Lithuania.

2.3. DNA Extraction, PCR, and Sequencing

Total DNA was extracted from insect remnants using the ammonium acetate extraction method [27]. The extracted DNA was then dissolved in 20 μL of 1× TE solution. For genetic analysis, we used a nested PCR protocol which amplifies a fragment of 479 bp of the cytochrome b (cytb) gene of the Haemoproteus and Plasmodium parasites [28,29]. All samples were evaluated by electrophoresis using 2 μL of PCR product in a 2% agarose gel. One negative control (nuclease-free water) and one positive control (a sample with a single infection of Plasmodium relictum cytb lineage GRW4) were used in every run.
DNA fragments of all PCR-positive samples were sequenced in both directions with the corresponding primers using a Big Dye Terminator V3.1 Cycle Sequencing Kit and ABI PRISMTM 3100 capillary sequencing robot (Applied Biosystems, Foster City, CA, USA). Electropherograms were analyzed using Geneious Prime 2022.2.1 for quality, identification of possible mixed infections (one peak for single infection, two or more peaks at the same position for mixed infections), and to create a contig sequence. Then, the contigs were analyzed and compared to other sequences using BLAST (Basic Local Alignment Search Tool) in the MalAvi database (http://130.235.244.92/Malavi/, accessed on 1 July 2022) to determine parasite lineages. The sequences with at least one base-pair of difference from already deposited sequences were considered as new lineages [30]. All sequences were deposited in the GenBank (accession numbers OP546062-OP546095) and MalAvi databases.
To confirm some biting midges species or identify PCR-positive females from the Culicoides obsoletus group, we used the primers LCO1490 and HCO2198, which amplify a fragment of cytochrome c oxidase subunit I (COI) of the mitochondrial DNA of insects [31]. PCR products were sequenced from the 3′ end with a Big Dye Terminator V3.1 Cycle Sequencing Kit and ABI PRISMTM 3100 capillary sequencing robot (Applied Biosystems, Foster City, CA, USA). Sequences were analyzed using BioEdit software, and obtained sequences were compared with other sequences using the BLAST on the GenBank. Identifications were considered for the sequences that presented similarity > 99%. Morphological identification was consistent with the PCR-based identification of the insects. These sequences were deposited in GenBank (accession numbers OP692758-OP692766).

2.4. Correlation between Culicoides Species and Haemosporidian Lineages

To understand the interactions between biting midges and their Haemoproteus parasites in Europe, studies published in the continent to date were used. To access these studies, we used the PubMed and Google Scholar databases; the search terms were: “Culicoides AND Haemosporida”, “Culicoides AND Haemosporidian”, “Culicoides AND Haemoproteus”, and “Haemoproteus AND vectors”. Only studies that used molecular methods and amplified a fragment of the cytb gene, available in the MalAvi database, were included in this analysis. Information on the vector species and parasite cytb gene lineages was used to create a database (Supplementary Table S1). For the studies that analyzed the samples using insect pools, each pool was considered as one sample.
Information for each Culicoides species containing the same Haemoproteus cytb lineage was summarized to obtain an interaction matrix, having as frequency of interaction consisting of the number of infected biting midges by a particular parasite lineage. A bipartite network and an adjacency matrix organized in modules of Haemoproteus lineages and Culicoides spp. were constructed in R 4.0.5 [32] using the bipartite package [33].

3. Results

3.1. Biting Midges and Parasite Diversity

In all, 580 parous Culicoides females belonging to 11 different species, all of which were reported in Lithuania, were collected and dissected (Table 1). The most abundant species were the Culicoides obsoletus group (31.9%), Culicoides kibunensis (19.7%), Culicoides festivipennis (15.3%), and Culicoides punctatus (11.9%). Haemosporidian parasite DNA was detected in 35 biting midges (6%), one being positive for Plasmodium DNA, and the remaining 34 biting midges positive for Haemoproteus DNA. Nine different genetic lineages of Haemoproteus were detected, including two new lineages, CULKIB02 and CULKIB03. Only one mixed infection was identified (Table 1).

3.2. Microscopic Analysis

Microscopic analysis of salivary gland preparations from the PCR-positive samples showed the presence of sporozoites in 17 samples (Figure 2), nine of them from C. segnis and eight from C. kibunensis (Table 1). Almost all C. segnis that were positive by PCR were also positive for sporozoites, except one. This is the first time that C. segnis has been confirmed as a competent vector for H. fringillae CCF3, H. majoris PHSIB1, H. asymmetricus TUPHI01, and H. minutus TURDUS2. This is also the first report of C. kibunensis being a competent vector for H. belopolskyi HIICT1.

3.3. Interation Network between Biting Midge Species and Haemoproteus Lineages

The interaction network between Culicoides-Haemoproteus lineages (Figure 3 and Figure 4) showed that, in nature, C. kibunensis is frequently associated with H. asymmetricus TUPHI01 and H. minutus TURDUS2. Additionally, H. asymmetricus TUPHI01 also presented a high number of interactions with C. segnis and C. pictipennis. Many Haemoproteus lineages were found interacting with only one biting midge species (e.g., H. fringillae CCF3 with C. segnis; and H. concavocentralis HAWF2 with C. circumscriptus). This might indicate a vector specialization by the Haemoproteus lineages encountered in the included studies. While H. minutus TURDUS2 was found interacting with ten different species of biting midges (Figure 4), showing low specificity in terms of vectors. On the other hand, most Culicoides species presented interactions with several lineages, suggesting that these insects are generalists in terms of parasite lineages. Culicoides kibunensis and C. segnis were the biting midge species with the highest number of interactions with different Haemoproteus parasites, with 13 each (Figure 3 and Figure 4).
Modularity analysis detected six different modules (Figure 3), indicating that the transmission of Haemoproteus parasites in Europe presents a compartmentalized pattern, with no clear nestedness observed. In other words, different species or groups of species of Culicoides are more likely to transmit certain groups of Haemoproteus lineages.

4. Discussion

The key results of this study are the detection of sporozoites of H. minutus TURDUS2, H. asymmetricus TUPHI01, H. majoris PHSIB1, and H. fringillae CCF3 in salivary gland preparations from C. segnis, as well as sporozoites of H. belopolskyi HIICT1 in the salivary gland preparation of C. kibunensis, showing that they are competent vectors of these Haemoproteus parasites. Culicoides kibunensis and C. segnis seem to play an important role as vectors of Haemoproteus parasites, being reported interacting with many Haemoproteus species and lineages (Figure 3 and Figure 4). On the other hand, some Haemoproteus parasites, e.g., H. asymmetricus TUPHI01, seem to be restricted to certain species of biting midges, such as C. segnis, C. pictipennis and C. kibunensis. Additionally, the number of interactions between C. kibunensis and Haemoproteus lineages described and commonly found in Turdidae birds (TURDUS2 and TUPHI01) was considerably high; this might indicate some feeding preference of C. kibunensis for Turdidae birds.
We found 11 different species of biting midges in our study (Table 1), all of them previously reported in Lithuania [34]. The most abundant species were biting midges from the C. obsoletus group and C. kibunensis, as previously reported in this country [3,5,6]. Other biting midge species seem to be rare, as is the case of C. fagineus, C. reconditus, C. albicans, C. fascipennis, C. circumscriptus, and C. newsteadi [3,5,6]. On the other hand, we reported the presence of C. deltus, which seems to be a rare biting midge species, and which was not recorded in the mentioned studies; even though this Culicoides species can be found all over Europe [25], it seems to be rare in the Eastern part of the continent [35]. Nevertheless, it is necessary to mention that, in some parts of Lithuania, C. impunctatus is the most abundant species [36]. This shows that even though several studies have been conducted in Lithuania, biting midge species diversity can vary from year to year, and between study sites, highlighting the importance of conducting more studies in different areas in the country to better understand the diversity of insects and their potential as vectors of Haemoproteus parasites.
The overall prevalence of Haemoproteus in Culicoides biting midges (5.9%) was similar to that noted in other studies conducted in Lithuania [3,5,6]. However, our results differed from other studies, with a prevalence higher than that in Bulgaria (approximately 2%) [8,9], as well as in Kaliningrad Oblast, Russia (1.7%) [7], but lower than that in Spain (13.4%) [10]. These differences should be carefully interpreted, since it depends on the study site, the density of insects, the diversity of the Culicoides species, their feeding preferences, the diversity of the bird species, the prevalence of Haemoproteus parasites in the bird populations, and the time of the year that the study was conducted.
Concerning the presence of Haemoproteus parasites in C. obsoletus group, the most abundant biting midge in the present study, we found a low prevalence of infections (only 1.1%). A study conducted in one of the areas that we sampled (Figure 1, area 1), did not report any infections in this biting midges species [6]. Interestingly, another study conducted in the same area (Figure 1, area 1) in 2016 reported a prevalence of approximately 6% [3], the same prevalence that was reported in the Curonian Spit, also in Lithuania [5]. This shows how dynamic infections can be in nature, and how much they can change between study sites and time of the year, increasing the complexity of understanding host-parasite-vector relationships. It is necessary to mention that, even though Haemoproteus DNA has been frequently found in C. obsoletus, showing that this biting midge species eventually feeds on birds, despite its mammalophilic behavior [17,19], Haemoproteus sporozoites have not been previously reported in this biting midge species.
The second-most abundant biting midge species, C. kibunensis, showed a high number of PCR-positive samples (14.9%), while a different positivity was previously reported in Lithuania, with 4.5% [3] and 45.5% [6] in Vilnius, and 7.8% in the Curonian Spit [5]. In the Czech Republic, the prevalence was of 51% of the insect pools analyzed [12], even though this insect species is distributed all over Europe [25]. In our study, C. kibunensis not only had a high number of PCR-positive females for Haemoproteus, but we also found sporozoites of H. belopolskyi HIICT1 in the salivary gland preparations (Table 1, Figure 2), confirming that this parasite can be naturally transmitted by C. kibunensis. This biting midge species was already reported to be a competent vector for H. pallidus PFC1, H. minutus TURDUS2, and H. asymmetricus TUPHI01 [3,6]. Culicoides kibunensis has a certain flexibility in host selection, and even though it was reported to feed mainly on mammals, it also takes blood meals from birds [18].
Culicoides festivipennis, another abundant biting midge species in our study, has been reported by several studies, including ours, to be positive for Haemoproteus DNA [3,5,8,12]. However, sporozoites were never found in salivary gland preparations. It might be that this biting midge species is a competent vector of Haemoproteus parasites, especially because of its ornithophilic feeding habits [19]. Furthermore, Haemoproteus infections have a relatively low prevalence in wild Culicoides [3,5,6,7,8,9], which means that a larger sampling might be necessary to prove the role of C. festivipennis as a natural vector of Haemoproteus parasites.
Although C. segnis was not one of the most abundant biting midge species in the present study, it showed a high positivity in the PCR for Haemoproteus and a high positivity for sporozoites in the salivary gland preparations (Table 1). The prevalence was similar to the one previously reported in the Curonian Spit [5]. So far, C. segnis has been reported to be positive for Haemoproteus DNA only in Lithuania and the Czech Republic [5,6,12], while only recently, it has been proven to be a competent vector of H. majoris CCF5 and H. tartakovskyi HAWF1 [5]. Our study adds three more species (four genetic lineages) of Haemoproteus to the list of species that C. segnis can transmit: H. minutus TURDUS2, H. asymmetricus TUPHI01, H. fringillae CCF3, and H. majoris PHSIB1.
For many years, a considerable number of experimental infections were performed with wild and laboratory-reared biting midges [4]. The most common experimental model is probably C. impunctatus [4], which was also collected and dissected in our study (Table 1). It has been shown to be a competent vector for at least 13 species of Haemoproteus parasites [4,16]. However, in this study all C. impunctatus were PCR-negative for Haemoproteus DNA, similar to the results reported in the literature, with no or a small prevalence of PCR-positive samples [6,7]. This means that, even though this biting midge species can transmit these parasites, the transmission probably does not occur in nature due to their mammalophilic habits. The fact that Haemoproteus DNA was found in those insects shows that they can sporadically feed on birds; however, to transmit Haemoproteus parasites, a second blood meal from a susceptible bird would be necessary (the sporozoite will take around seven days to develop after the first blood meal from an infected bird), and this might be very unlikely in nature. Additionally, these experiments were conducted with infected birds being held by hand, allowing biting midges to feed naturally [13,37]. The fact that C. impunctatus is highly attracted to humans [38,39] might induce some biased results, which would not reflect the natural transmission dynamics, even though proving that the biting midge can support parasite development, and it might not be a specialist at the vector level.
Another important characteristics of C. impunctatus that can directly affect Haemoproteus transmission is the fact that this biting midge species is considered to be bivoltine (having two generations per year) and autogenous (the first batch of eggs is produced without a blood meal) [38,40]. After laying their first batch of eggs, C. impunctatus females would become responsive to animal bait and to light, and if the biting midge can have a blood meal, then a second batch of eggs could be laid [41]. Due to vector biology, a third batch of eggs would be unlike, meaning that C. impunctatus females would not feed on a vertebrate host again and, even though they are infected by Haemoproteus parasites, they would not transmit it. However, there are only a few studies concerning this topic with regards to C. impunctatus [38,41]. Thus, the lack of natural infections in this biting midge species in nature is probably the result of a combination of these features: mammalophilic habits, bivoltinism, and autogeny.
The interaction network analysis confirms this complex relationship between the parasites and their vectors, showing an intricate pattern (Figure 3 and Figure 4). Our analysis showed that C. kibunensis plays an important role in the transmission of Haemoproteus parasites in Europe. Not only it was found interacting with several Haemoproteus lineages (13 in total), but it also represented the biting midge species with the highest diversity of Haemoproteus lineages in the present study. However, its main interactions were with H. minutus TURDUS2 and H. asymmetricus TUPHI01, both lineages were described in Turdus merula and Turdus philomelos, respectively; even though these lineages were reported in other bird species, their main hosts are Turdidae birds.
Culicoides segnis also presented a high number of interactions with Haemoproteus lineages (also 13), highlighting the interactions with H. asymmetricus TUPHI01. This might indicate that C. segnis also plays an important role in the transmission of certain Haemoproteus parasites in nature, even though sporozoites were only recently found in their salivary glands. It is likely that C. segnis has a broader range of feeding preference in terms of host species.
It is necessary to mention that C. circumscriptus also had a considerably high number of interactions with different Haemoproteus species (ten in total). This is also the only biting midge species that was found to be positive for Haemoproteus DNA lineages found in birds belonging to Strigidae (owls), Accipitridae (hawks), and Corvidae (crows and ravens) (Figure 3 and Figure 4), according to the MalAvi database (accessed on September 2022 [30]). This might be explained by the fact that some species of Culicoides prefer to live in places at different heights [19,42]. Following this, C. circumscriptus was more frequently sampled at 20–26 m above the ground [43], were most species of the mentioned families live; while C. kibunensis was caught in higher numbers at ground level [42], where most Turdidae birds the majority of the day.
The fact that different biting midge species prefer different heights is one explanation for why we noticed those differences in the network interaction. This shows that the transmission of vector-borne diseases is complex and can be influenced by several different factors. First, feeding preference should be considered, since some biting midge species are mammalophilic and even though they sporadically feed on birds, they probably do not play an important role in Haemoproteus transmission. Second, the capacity of the parasite to influence the vector behavior, which was investigated for some avian Plasmodium parasites, which are closely related to Haemoproteus, and Culex mosquitoes [44,45,46,47,48]. However, such research has never been conducted using a Culicoides-Haemoproteus system. Third, and probably one of the main factors that should be taken into account when investigating vector-borne diseases, is the need to have an infected host, a competent vector, and a susceptible host in the same place and at the same time.
It is necessary to mention that all the parasites found in the present study infect birds (Table 1) [1]. This allowed us to access the information regarding all Culicoides that were PCR-positive for Plasmodium/Haemoproteus fed on birds, even though they did not have sporozoites in their salivary glands. This information is important, due to the lack of research on Culicoides feeding preferences.
In our study, one sample was positive for avian Plasmodium DNA, even though these parasites are not transmitted by Culicoides insects. This is not new, and has been frequently reported in the literature [3,7,8,10], indicating that the biting midge fed on an infected bird. However, since the parasite does not complete its development in biting midges, they cannot be considered vectors of Plasmodium parasites.

5. Conclusions

Culicoides segnis is a competent vector for H. minutus TURDUS2, H. asymmetricus TUPHI01, H. majoris PHSIB1, and H. fringillae CCF3; and C. kibunensis is a competent vector for H. belopolskyi HIICT1. We are adding two new Haemoproteus lineages to the list of parasites transmitted in Europe and highlighting the important role that C. segnis and C. kibunensis play in the transmission of these parasites. The relationship between Culicoides biting midges and Haemoproteus parasites is complex and represents a challenge regarding understanding how these parasites are transmitted in nature. More studies focusing on insect biology and the identification of the natural vectors of Haemoproteus parasites (combining molecular tools and the investigation of sporozoites in salivary gland preparations) should be encouraged.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/d14110957/s1, Table S1: Studies in Europe investigating the presence of Haemoproteus parasites DNA in wild Culicoides biting midges used in the interaction network analysis.

Author Contributions

Conceptualization, C.R.F.C.; methodology, R.B.; formal analysis, C.R.F.C. and C.H.-L.; investigation, C.R.F.C., C.H.-L., M.D., K.V.-P., and R.B.; resources, C.R.F.C. and R.B.; data curation, C.R.F.C. and C.H.-L.; writing—original draft preparation, C.R.F.C. and C.H.-L.; writing—review and editing, C.R.F.C., C.H.-L., M.D., K.V.-P. and R.B.; supervision, C.R.F.C.; project administration, C.R.F.C.; funding acquisition, C.R.F.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Research Council of Lithuania (No. S-MIP-21-55, Carolina Romeiro Fernandes Chagas).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the Supplementary Material, in the GenBank database (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/genbank/ (accessed on 1 September 2022)-accession numbers OP546062-OP546095, OP692758-OP692766), and in the MalAvi database (http://130.235.244.92/Malavi/, accessed on 1 August 2022).

Acknowledgments

The authors would like to thank the staff of the Vilnius University Botanical Garden for allowing us to set the traps, and to Ervin Komar for assistance in Culicoides trapping. We also would like to thank Alžbeta Šujanová and Nathan Jay Baker for their valuable discussions. The graphical abstract was created with BioRender.com.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Diversity 14 00957 g001 550
Figure 1. Collection sites in Lithuania: Verkiai Regional Park (1), Vilnius University Botanical Garden (2), Puvočiai (3 and 4), Ventės Ragas (5), and its surroundings (6). Produced with Google Earth Pro v. 7.3.6.9277.
Figure 1. Collection sites in Lithuania: Verkiai Regional Park (1), Vilnius University Botanical Garden (2), Puvočiai (3 and 4), Ventės Ragas (5), and its surroundings (6). Produced with Google Earth Pro v. 7.3.6.9277.
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Figure 2. Sporozoites of Haemoproteus asymmetricus cytochrome b lineage TUPHI01 (A,C), H. fringillae CCF3 (B) in Culicoides segnis, and H. minutus TURDUS2 in Culicoides kibunensis (D) in salivary gland preparations. Scale bar: 10 μm. Methanol-fixed and Giemsa-stained.
Figure 2. Sporozoites of Haemoproteus asymmetricus cytochrome b lineage TUPHI01 (A,C), H. fringillae CCF3 (B) in Culicoides segnis, and H. minutus TURDUS2 in Culicoides kibunensis (D) in salivary gland preparations. Scale bar: 10 μm. Methanol-fixed and Giemsa-stained.
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Figure 3. Modularity interaction network between Culicoides species (rows) and Haemoproteus lineages (columns). The blue squares represent the interactions between insects and parasites; the darker the square, the more often these interactions were registered. Culicoides species and Haemoproteus lineage investigated and found in this study are marked in bold. Red lines are separating different modules.
Figure 3. Modularity interaction network between Culicoides species (rows) and Haemoproteus lineages (columns). The blue squares represent the interactions between insects and parasites; the darker the square, the more often these interactions were registered. Culicoides species and Haemoproteus lineage investigated and found in this study are marked in bold. Red lines are separating different modules.
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Figure 4. Bipartite interaction network between Haemoproteus cytb lineages (top, in blue boxes) and Culicoides species (bottom, in light red boxes). The width of the boxes is proportional to the frequency of the occurrence of a particular taxon. The width of connecting lines is proportional to the number of interactions observed between each Culicoides species and Haemoproteus lineage. Culicoides species and Haemoproteus lineages investigated and found in this study are marked in bold.
Figure 4. Bipartite interaction network between Haemoproteus cytb lineages (top, in blue boxes) and Culicoides species (bottom, in light red boxes). The width of the boxes is proportional to the frequency of the occurrence of a particular taxon. The width of connecting lines is proportional to the number of interactions observed between each Culicoides species and Haemoproteus lineage. Culicoides species and Haemoproteus lineages investigated and found in this study are marked in bold.
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Table
Table 1. Summary of collected Culicoides biting midges, with their respective Haemoproteus species and lineages.
Table 1. Summary of collected Culicoides biting midges, with their respective Haemoproteus species and lineages.
Culicoides Speciesn (Prevalence)Haemosporidian
cytb Lineage		Parasite Species
(No. of PCR-Positive Insects)
C. chiopterus2 (0)--
C. deltus1 (0)--
C. festivipennis89 (4.5)HIICT1
SYAT05		Haemoproteus belopolskyi (3)
Plasmodium vaughani (1)
C. impunctatus53 (0)--
C. kibunensis114 (14.9)HIICT1
CULKIB02
CULKIB03
TUCHR01
TUPHI01
TURDUS2		Haemoproteus belopolskyi (4) 1
Haemoproteus sp. (1)
Haemoproteus sp. (1)
Haemoproteus minutus (1)
Haemoproteus asymmetricus (4) 2
Haemoproteus minutus (6) 3
C. obsoletus group *185 (1.1)HIICT1
WW2		Haemoproteus belopolskyi (2)
Haemoproteus majoris (1)
C. pallidicornis18 (5.3)HIICT1Haemoproteus belopolskyi (1)
C. pictipennis2 (50)TUPHI01Haemoproteus asymmetricus (1)
C. punctatus69 (0)--
C. reconditus1 (0)--
C. segnis42 (23.8)CCF3
mix infection
PHSIB1
TUPHI01
TURDUS2		Haemoproteus fringillae (2) 1
Haemoproteus spp. (1) 1
Haemoproteus majoris (1) 1
Haemoproteus asymmetricus (4) 4
Haemoproteus minutus (2) 4
Culicoides sp.3 (0)--
n = number of investigated insects. Bold indicates the Haemoproteus species from which sporozoites were found in salivary gland preparations. 1 one sample positive for sporozoites; 2 two samples positive for sporozoites; 3 five samples positive for sporozoites; 4 all samples were positive for sporozoites. (*) positive insects were molecularly confirmed to be C. obsoletus. (-) not evaluated.
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Chagas, C.R.F.; Hernández-Lara, C.; Duc, M.; Valavičiūtė-Pocienė, K.; Bernotienė, R. What Can Haemosporidian Lineages Found in Culicoides Biting Midges Tell Us about Their Feeding Preferences? Diversity 2022, 14, 957. https://0-doi-org.brum.beds.ac.uk/10.3390/d14110957

AMA Style

Chagas CRF, Hernández-Lara C, Duc M, Valavičiūtė-Pocienė K, Bernotienė R. What Can Haemosporidian Lineages Found in Culicoides Biting Midges Tell Us about Their Feeding Preferences? Diversity. 2022; 14(11):957. https://0-doi-org.brum.beds.ac.uk/10.3390/d14110957

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Chagas, Carolina Romeiro Fernandes, Carolina Hernández-Lara, Mélanie Duc, Kristina Valavičiūtė-Pocienė, and Rasa Bernotienė. 2022. "What Can Haemosporidian Lineages Found in Culicoides Biting Midges Tell Us about Their Feeding Preferences?" Diversity 14, no. 11: 957. https://0-doi-org.brum.beds.ac.uk/10.3390/d14110957

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