The development of avian malaria co-infections in experimentally exposed vectors

The development of avian malaria co-infections in experimentally exposed vectors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The development of avian malaria co-infections in experimentally exposed vectors Rita Žiegytė, Rasa Bernotienė, Vaidas Palinauskas This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9368372/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Co-infections with multiple parasites are widespread in wild populations and can influence both host-parasite and vector-parasite interactions. Among haemosporidian parasites, co-infections involving different genera or occasionally, multiple species within the same genus – such as Plasmodium – have been documented in birds. However, the impact of such co-infections on sporogonic development within vectors remains poorly understood. Methods This study investigated the sporogony, transmission and virulence of single and co-infections of Plasmodium relictum (genetic lineage pSGS1) and Plasmodium elongatum (genetic lineage pERIRUB1) in Culex pipiens pipiens form molestus mosquitoes, using microscopic examination and PCR testing. Mosquitoes were experimentally infected by allowing them to take a blood meal from canaries carrying either a parasite of a single species or exposed to birds infected with two species, resulting in double infection. After blood meals, the insects were maintained until the parasite reached the sporozoite stage. Results Our findings reveal that both parasite species can complete sporogonic development within the same insect vector and can be simultaneously transmitted to a healthy avian host through a single mosquito bite. Additionally, the study shows distinct effects of single and co-infections on mosquito survival, demonstrating specific interactions between parasites during sporogonic development in the vector. Co-infected mosquitoes experienced lower mortality than those infected with a single P. elongatum infection. This could be due to a potential suppressive effect of P. relictum on the development of P. elongatum within the mosquito, thereby reducing harm to the vector. Conclusions This study deepens our understanding of avian malaria co-infections by focusing on co-infections within vectors, and highlights their potential impact on transmission efficiency, vector fitness and the ecological dynamics of disease spread. Culex pipiens co-infections mosquitoes Plasmodium relictum Plasmodium elongatum sporozoites Figures Figure 1 Figure 2 Background Co-infections involving multiple haemosporidian and other parasite species are frequently observed in natural populations of humans, birds, and other vertebrates [1−8]. Interactions between co-infecting parasites can significantly influence disease severity, parasite development, transmission success, and host survival [ 2 , 7 , 8 ]. These interactions may be suppressive, facilitative, or neutral, depending on the parasite species or genetic lineages involved [9−12]. One key factor shaping co-infection outcomes is parasite competitiveness, which determines infection dynamics and transmission potential within hosts and vectors [ 13 ]. Among avian malaria parasites, Plasmodium relictum and Plasmodium elongatum are two widespread and highly studied species. Plasmodium relictum , first described by Grassi et al. [ 14 ], is one of the most extensively investigated avian malaria parasites. Several mitochondrial DNA cytochrome b ( cyt b) gene lineages (e.g., pSGS1, pGRW11, pGRW4, pLZFUS01, pPHCOL01) attributed to this species are deposited in GenBank and MalAvi database ( http://mbio-serv2.mbioekol.lu.se/Malavi ) [ 15 , 16 ]. This parasite is globally distributed and recognized as an invasive pathogen responsible for severe disease outbreaks and population declines, particularly in immunologically naïve bird species [1−25]. The primary vectors of P. relictum are mosquitoes of the genus Culex [ 26 ]. Plasmodium elongatum is another pathogenic avian malaria parasite that comprises several genetic lineages, including pGRW6 and pERIRUB01. The pGRW6 lineage is particularly virulent to non-adapted bird species, such as brown kiwi and different species of penguins [ 21 , 27 ]. Similar to P. relictum , this parasite is mainly transmitted by Culex mosquitoes [ 28 ]. Experimental studies have demonstrated that the parasite of pERIRUB01 lineage is capable of completing sporogonic development, including sporozoite formation, in Culex quinquefasciatus and C. p. pipiens f. molestus mosquitoes [ 29 ]. Experimental studies in avian hosts have demonstrated that outcomes of co-infections involving P. relictum depend on the specific combination of parasite lineages. For example, co-infection with closely related P. relictum lineages pGRW11 and pSGS1 does not alter overall parasitemia or increase virulence compared with single infections [ 8 ]. Experimental infections of Culex p. pipiens f. molestus mosquitoes with these lineages showed successful sporozoite development for both parasites, although oocyst burden differed between genetic lineages [ 30 ]. In contrast, co-infection involving pSGS1 and P. relictum lineage pGRW4 distributed in Africa resulted in transient infection in birds, with pGRW4 being suppressed and cleared from peripheral blood [ 31 ]. This finding suggest that locally dominant parasite lineages may competitively exclude less-adapted lineages, thereby influencing transmission success. Conversely, some interactions may be facilitative rather than competitive. For instance, the presence of the P. relictum pSGS1 was shown to enhance parasitemia of the P. elongatum lineage pERIRUB01 without affecting its own development, resulting in co-infection virulence similar to that of the more pathogenic parasite [ 12 ]. Competitive interactions have also been documented among parasites of the related genus Haemoproteus in in vitro experimental studies [32−34]. These examples demonstrate the complexity of parasite interactions during co-infection, which vary depending on parasite species, lineages, and their competitive abilities. Co-infections involving Plasmodium species are also common in humans and their Anopheles mosquito vectors [ 4 ]. For instance, sporozoites of Plasmodium knowlesi, Plasmodium falciparum , and Plasmodium vivax were detected in Anopheles dirus mosquitoes [ 35 ]. However, it remains unclear whether such interactions originate from a single blood meal containing multiple parasite species or from multiple feeding events. It is still uncertain whether transmission of different Plasmodium species can occur through a single mosquito or require multiple infeced vectors carrying different parasite species [ 4 , 35 ]. Furthermore, it is unclear whether competition for red blood cells occurs during co-infections, and if so, what the implications of such interaction may be McQueen and McKenzie [ 9 ]. What is the effect of co-infections involving several Plasmodium species on gametocyte formation? [ 7 ]. These questions also arise in the context of human malaria research. Despite the immense relevance of research on human malaria, experimental investigations of co-infection dynamics are limited due to ethical constraints on experimental infections in vertebrate hosts. In contrast, avian malaria system provides valuable experimental model that allow controlled investigation of parasite-parasite interactions in vivo . As research into avian malaria progresses, more and more information is becoming available on the interactions between parasites in co-infected vertebrate hosts. However, experimental studies of these infections in vectors are still rare despite being highly relevant [ 4 , 19 , 26 , 27 ]. Many investigations report only partial sporogonic stages, such as ookinetes or oocysts [ 28 ] highlighting the persistent lack of comprehensive experimental evidence on the complete development of parasites in the vectors [ 26 ]. Based on previous experimental findings and in an effort to supplement existing knowledge about parasite development in vectors we hypothesize that co-infection with P. relictum and P. elongatum in Culex p . pipiens f. molestus mosquitoes will result in parallel development of both parasites to the sporozoite stage, potentially enabling their transmission during a single mosquito bite. The primary aim of this study is therefore to investigate the development, transmission potential, and virulence of P. relictum and P. elongatum during co-infection in Culex p. pipiens f. molestus mosquitoes, and to determine whether both parasites can be simultaneously transmitted to a healthy avian host by a single vector. By examining sporogonic stages in vectors, we aim to clarify whether such co-infections require multiple vector transmission events or can occur through a single mosquito. This experimental study contributes to a broader understanding of the development, transmission strategies, and ecological implications of haemosporidian co-infections. Methods Plasmodium parasites and experimental setup The study was performed at the State Scientific Research Institute Nature Research Centre (NRC) in Vilnius, Lithuania. Domestic canaries ( Serinus canaria domestica) , obtained from a commercial supplier, were used in the experiments. Prior to infection, all birds were confirmed to be uninfected with malarial parasites by both microscopic examination and PCR testing. To multiply Plasmodium parasites, birds were experimentally infected by intramuscular inoculation of infected blood into the pectoral muscles (Fig. 1 , Step 1), as described by Palinauskas et al. [ 20 ]. We used two avian malaria parasites – P. relictum pSGS1 isolated from a naturally infected Common crossbill ( Loxia curvirostra ) and P. elongatum pERIRUB01 isolated from a naturally infected European robin ( Erithacus rubecula ). Parasites were multiplied and subsequently cryopreserved in liquid nitrogen, following the protocol described by [ 36 ]. For the infection of the donor birds we used parasites of the 3rd passage after original isolation of P. relictum and P. elongatum . We used four groups of donor birds for the experiment: birds infected with P. relictum pSGS1 (group B1); birds infected with P. elongatum pERIRUB1 (group B2); birds co-infected with P. relictum and P. elongatum , pSGS1×pERIRUB1 (group B3); and uninfected control birds (group B4). Each group consisted of 6 canaries housed in individual cages in a vector-free room under controlled environmental conditions (10:14 h light/dark photoperiod, temperature 21 ± 1° C). Plasmodium relictum was identified following the description by Valkiūnas [ 19 ] while P. elongatum was identified as described in Valkiūnas [ 19 ] and Palinauskas et al. [ 29 ]. For parasitological examination, approximately 100 fields were studied at high magnification (1000×). Intensity of parasitemia was estimated as a percentage 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. [ 37 ]. We used an Olympus B51 light microscope (Olympus, Japan) with a digital camera and Olympus DP-SOFT software to analyse blood slides. The presence of a single and co-infection was confirmed by microscopic examination of blood smears and further confirmed by PCR-based methods and sequencing parasite’s DNA (see details below). Co-infections were identified by visual inspection of electropherograms based on double base-calling, as described by Pèrez-Tris and Bensch [ 38 ]. After the development of parasitemia in the blood of infected birds, they were used as donors to infect Culex p. pipiens f. molestus mosquitoes (Fig. 1 , Step 2) as described below. Gametocytemia was assessed in all donor canaries immediately after mosquitoes had taken a blood meal. Experimental infections and dissection of mosquitoes for sporogony investigation Culex p. pipiens f. molestus mosquitoes, reared in the laboratory at the NRC, were experimentally infected with Plasmodium parasites. Four groups of mosquitoes were formed based on their donor birds: mosquitoes fed on B1 group birds infected with P. relictum pSGS1 (group M1); mosquitoes fed on B2 group birds infected with P. elongatum pERIRUB1 (group M2); mosquitoes fed on B3 group birds co-infected with P. relictum and P. elongatum pSGS1×pERIRUB1 (group M3); and mosquitoes fed on uninfected control birds (group M4). Mosquitoes were fed as described by Žiegytė et al. [ 30 ] and Kazlauskienė et al. [ 39 ]. Briefly, infected canaries exhibiting gametocytemia were placed in a tube within an experimental mosquito cage, with only their legs exposed to mosquito bites. Blood feeding was allowed to proceed for approximately 10 minutes to 1 hour. Fully engorged female mosquitoes were taken from the cages using an aspirator, transferred to individual small insect cages, and maintained under controlled conditions (temperature 23° C, 60 ± 2% relative humidity, and 16:8 h L/D photoperiod). All experiments were conducted in parallel. Dissection and preparation of permanent preparations for identifying sporogonic stages in mosquitoes (ookinetes, oocysts, and sporozoites), as well as their staining techniques, were carried out following established protocols [ 19 , 29 , 30 , 39 ]. Thoraxes of dissected mosquitoes were preserved in 96% ethanol for subsequent PCR-based confirmation of Plasmodium lineages (see details below). Infection of canaries by bites of experimentally infected mosquitoes To evaluate the infectivity and the transmission potential of Plasmodium sporozoites developed in mosquitoes with co-infections of P. relictum and P. elongatum , uninfected domestic canaries were exposed to bites of experimentally infected mosquitoes (Fig. 1 , Step 3). As recipients for the bites we used 5 healthy canaries as detected using both using microscopy and PCR. Individual mosquitoes, later confirmed to carry sporozoites in their salivary glands, were allowed to take a blood meal by placing the bird’s legs into a mesh-covered feeding cage for approximately 10−15 minutes as described above. Only one mosquito was allowed to feed on each bird to ensure that any subsequent infection could be traced to a single transmission event. Immediately after feeding, the mosquito was dissected, and its salivary glands were removed for microscopic preparations to determine the presence of sporozoites. Blood samples were collected from the exposed canaries at 4, 8, 12, 16, 20, and 24 days post infection (dpi) and examined by both microscopy and PCR to identify parasite species of developed blood stages. PCR-based examination, sequencing and statistical analysis Total DNA was extracted from samples (birds’ blood stored in SET buffer and mosquitoes placed in absolute ethanol) using an ammonium acetate extraction method [ 40 ]. To identify parasites, we used a nested PCR protocol [ 41 , 42 ]. We amplified a fragment of the cyt b gene using initial primers HaemNFI and HaemNR3, which amplify DNA fragments of haemosporidians belonging to Haemoproteus, Plasmodium and Leucocytozoon [ 42 ] and inner primers HaemF and HaemR2, which are specific to Haemoproteus and Plasmodium spp. [ 41 ]. The amplification was evaluated by running 3 µl of the final PCR product on a 1,5% agarose gel. One negative control (nuclease-free water) and one positive control ( P. relictum pSGS1 microscopy positive blood) were used for every 14 samples to control for false amplifications. Fragments of DNA from the positive samples were sequenced using a Big Dye Terminator V3.1. Cycle Sequencing kit and an ABI PRISMTM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed using BioEdit software and compared with sequences deposited in the Malavi database [ 15 ]. The presence of a single infection and co-infection was confirmed by visual inspection of double-base-calling at specific sequence sites on the electropherogram [ 38 ]. We used a chi-square (χ 2) test to assess differences between numbers of surved and dead mosquitoes. Results Plasmodium parasites in donor birds Microscopic examination of gametocytemia in donor birds revealed intensities of 0.05% in group B1 infected with single P. relictum ; 0.18% in group B2 infected with single P. elongatum ; and 0.43% (0.09% P. relictum and 0.34% P. elongatum ) in group B3 with co-infection. No parasites were detected in control group B4. These birds were subsequently used as donors for experimental mosquito infections. Suitable gametocytemia for healthy mosquitoes to infect birds developed approximately 8–14 days after the parasites were inoculated into the birds. Sporogony and virulence in mosquitoes A total of 269 Culex p. pipiens f. molestus mosquitoes were experimentally infected: 75 mosquitoes were infected with P. relictum (M1), 98 − with P. elongatum (M2), 56 − with co-infection (M3), and 40 mosquitoes served as uninfected controls (M4) (Table 1 ). Table 1 The virulence of Plasmodium infections in Culex p. pipiens f. molestus mosquitoes. Experimental group No. of infected mosquitoes No. of dead mosquitoes Mortality rate (%) M1 ( P. relictum ) 75 34 45.3 M2 ( P. elongatum ) 98 62 63.3 M3 (co-infection) 56 19 33.9 M4 (uninfected mosquitoes) 40 16 40.0 Sporogonic development in M1 group was completed in 37 (90.2%) of 41 surviving mosquitoes between 20 and 23 days post exposure (dpe) with sporozoites observed in the salivary glands. For group M2, sporogony was completed in 14 (38.9%) of 36 surviving mosquitoes between 20 and 25 dpe. In the co-infection group M3, 15 (40.5%) of 37 surviving mosquitoes completed sporogony for both parasites within the same 20–25 dpe period. The presence of single and co-infection of P. relictum and P. elongatum in salivary glands was confirmed using PCR and sequencing by visual inspection of double-base-calling in specific locuses in sequence electropherograms. Even though the sporozoite timing was similar across all experimental groups, the mortality rates of mosquitoes varied. The development of a single P. relictum (M1) did not result in a higher mortality rate among the exposed insects (34 out of 75, or 45.3%) compared with the control group (40.0%) until 20 dpe (p = 0.6) (Table 1 ). In contrast, the mortality of mosquitoes infected with P. elongatum (M2) was significantly higher, reaching 63.3%, with a significant difference in mortality between this group and the group M1 (p = 0.02, χ² = 5.53) as well as the M3 group, infected with both parasites (p = 0.0005, χ² = 12.3) (Table 1 ). Mortality of the M2 group did not differ significantly from that of the control group M4 (p = 0.4). Similarly, mortality of the M3 group did not differ from that of the control group M4 (p = 0.7). Transmission of Plasmodium parasites to birds via a single mosquito bite To evaluate the infectivity of co-infection-derived sporozoites, mosquitoes with co-infection (M3) were allowed to take a blood meal from healthy canaries. Successful development of single infections of investigated parasites in Culex p. pipiens f. molestus mosquitoes fed on experimentally infected canaries was proved in our previous studies [ 29 , 30 ], so in order to reduce the number of animals used for experimental procedures, we did the experiment of the final transmission only with co-infected mosquitoes. The co-infected mosquitoes from group M3 were allowed to feed on the blood of 5 healthy canaries, with a single mosquito feeding on the blood of a single canary. The mosquitoes were immediately dissected after the blood meal, and sporozoites were detected in their salivary glands. In two of the five canaries, parasites of both Plasmodium species developed, and the birds became co-infected, as determined by microscopy and PCR-based methods (Fig. 2 ). Sequencing confirmed the presence of both P. relictum pSGS1 and P. elongatum pERIRUB1 lineages in two experimentally infected birds, as evidenced by double base calls in DNA sequencing chromatograms at diagnostic nucleotide positions where the two sequences differ. In one double infected canary, only P. relictum parasite successfully developed, while the other two canaries remained uninfected, despite the detection of parasite sporozoites in mosquitoes that had fed on them. Between 8 and 16 dpi, parasites of both P. relictum and P. elongatum were identified in blood smears from co-infected birds (Fig. 2 ). Discussion This study provides novel insights into the development and transmission potential of two distantly related avian malaria parasites, P. relictum and P. elongatum , during co-infection in C. p. pipiens f. molestus mosquitoes. Our results reveal that both parasite species can complete sporogonic development within the same vector and can be simultaneously transmitted to a naïve avian host through a single mosquito bite. Additionally, the study shows distinct effects of single and co-infections on mosquito survival, suggesting that parasite interactions during sporogony may influence vector fitness. Sporozoites of both P. relictum and P. elongatum were detected in the salivary glands of infected mosquitoes between 15 and 21 dpe. These results are consistent with previous studies showing that P. relictum completes sporogonic development in Culex p. pipiens f. molestus mosquitoes within approximately 14–32 dpe, with sporozoites commonly observed in exposed insects [ 30 , 39 ]. Although P. relictum is capable of completing sporogony in more than 25 mosquito species belonging to different genera, mosquitoes of the genus Culex are considered its primary vectors [19,26−28]. For P. elongatum (pERIRUB01), sporozoites were observed between 18 and 23 dpe in this study, which is comparable to earlier experimental findings reporting sporozoite formation between 20 and 22 dpe in Culex p. pipiens f. molestus , where sporozoites were detected in approximately 13–20% of exposed mosquitoes [ 29 ]. Historically, attempts to identify P. elongatum vectors involved several mosquito genera, including Aedes, Anopheles, Culex , and Culiseta , but many of these early studies produced inconclusive results [ 19 , 28 ]. The observed differences in mosquito mortality among infection groups demonstrate substantial variation in vector tolerance depending on infecting Plasmodium species. Mosquitoes infected with P. relictum alone did not exhibit elevated mortality compared with uninfected controls. This observation is consistent with previous studies suggesting that the pSGS1 lineage of P. relictum may cause relatively low virulence in some vertebrate hosts [ 12 , 20 ]. In contrast, mosquitoes infected with P. elongatum alone experienced significantly higher mortality, a pattern that aligns with the high pathogenicity of this parasite in avian host [ 12 , 29 ]. Interestingly, mosquitoes co-infected with both parasites showed lower mortality than those infected with P. elongatum alone. This pattern may indicate a suppressive interaction, whereby P. relictum partially limits the development of P. elongatum within the mosquito. Such suppression could reduce physiological stress on the vector and thereby improve survival. However, further targeted experiments are necessary to determine whether this effect reflects direct parasite competition, immune-mediated interactions, or resource limitation within the mosquito. Vector mortality is sometimes associated with the intensity of parasitaemia, i.e. parasite burden in the blood of infected birds, as demonstrated for Haemoproteus parasites [ 43 – 45 ]. In the present study, however, mosquito mortality did not correlate with parasite gametocytemia in donor birds. The highest gametocitemia occurred in birds with co-infection, reaching 0.43%, and mosquitoes in this group showed the lowest mortality (33.9%), comparable to the control group. In contrast, the highest mortality (63.2%) was observed in the group infected with single P. elongatum , where the donor bird exhibited only 0.18% gametocytemia. Previous studies suggest that high parasite burdens may reduce mosquito survival, potentially making parasite transmission more efficient during the chronic rather than acute infection stage [ 46 ]. It is notable that co-infections involving P. relictum and P. elongatum are known to be highly virulent and often lethal in birds [ 12 ], whereas in mosquitoes the same parasite combination appears less harmful than infection with single P. elongatum . This contrast highlights the complexity of host-parasite-vector interactions and indicates that the fitness costs of co-infections may differ substantially between vertebrate and invertebrate hosts. Complex interactions between co-infecting haemosporidian parasites have also been demonstrated in experimental studies of Haemoproteus parasites. In vitro experiments have shown complete inhibition of ookinete development between certain parasite species, such as Haemoproteus pallidus and Haemoproteus minutus [ 32 , 33 ], and between H. pallidus and Haemoproteus tartakovskyi [ 33 ]. Conversely, the experiments with H. tartakovskyi and Haemoproteus parabelopolskyi have demonstrated increased reproductive activity and a higher number of normaly forming ookinetes of the first parasite, but the blockage of ookinete development of H. tartakovskyi [ 34 ]. These examples show that parasite-parasite interactions during sexual reproduction can range from strong competition to facilitation and may substantially influence transmission success. The most significant finding of this study is the successful transmission of two genetically distant Plasmodium species from a single co-infected mosquito to a naïve canary host during a single feeding event. To our knowledge, this is the first experimental demonstration of simultaneous transmission of two avian malaria parasites by a single mosquito bite. Historical literature shows that co-infections of Plasmodium species are common in human malaria systems [ 4 , 7 , 47 – 49 ]. For example, infections with P. vivax frequently occur following P. falciparum malaria episodes in approximately one-third of cases [ 4 ]. However, available entomological evidence suggests that most of these co-infections likely result from sequential inoculation events by different mosquitoes rather than from a single mosquito bite [ 4 ].Our experimental results indicate that simultaneous transmission by a single vector is nevertheless possible. Similar patterns may occur in other malaria systems. Holzschuh et al. [ 7 ] reported that Plasmodium malariae is detected more frequently in individuals co-infected with P. falciparum − of the four parasite species P . vivax , P . malariae , and P . ovale , that were circulating simultaneously, P. malaria benefited the most from being co-infected with the others, suggesting that interactions between parasite species may facilitate the persistence or spread of certain parasites. Obtained gametocytes of both species with a single blood meal should result, at least in some cases, in the transmission of both parasite species during a single mosquito bite. Such assumptions could be made based on our experimental data; however, there is a possibility that these specific parasites may inhibit sporogonic development in the vector, even if they both can persist simultaneously in the blood stream of the vetebare host. Similarly, depending on their relative reproduction rates and the timing of inoculation, one species may suppress the other when competing for red blood cells [ 9 ]. Although co-infections are widely documented in wild bird and human populations [ 3 , 7 , 10 , 11 , 26 , 49 , 50 ], they are generally assumed to result from sequential bites by multiple infected mosquitoes or from long-term parasite persistence within the host. The demonstration that P. relictum and P. elongatum can be transmitted simultaneously by a single mosquito challenges this assumption and suggests that single vector-mediated transmission may contribute more strongly to mixed infecions than previously recognized. The capacity of a single mosquito to transmit multiple parasite species may influence within-host parasite competition, host immune responses, interactions with the host microbiome, and the structure of parasite communities. Such transmission events may facilitate the establishment of mixed infections and potentially alter disease outcomes in avian hosts [ 5 , 12 ]. Furthermore, vectors harboring multiple Plasmodium lineages may serve as environments where genetic recombination can occur, potentially increasing parasite genetic diversity and adaptive potential [ 51 ]. Understanding the dynamics of parasite interactions within vectors is an understudied aspect of malaria research. Molecular approaches such as quantitative PCR ( q PCR) could help address this gap by enabling precise quantification of each parasite species abundance during sporogonic development. Applying such technique to various combinations of Plasmodium species would substantially advance our understanding of parasite competition in mosquitoes and transmission dynamics. Conclusions This study demonstrates that co-infection with P. relictum and P. elongatum can successfully complete sporogonic development to infective sporozoite stages in C. p. pipiens f. molestus mosquitoes. Sporozoites of both parasite species were detected in mosquito salivary grands between 21 and 23 dpe and both parasites were simultaneously transmitted to a naïve avian host through a single mosquito bite. Mosquitoes infected with both parasite species exhibited lower mortality than those infected with P. elongatum alone, suggesting a potential suppressive interaction between parasites within the vector. However, the mechanisms underlying this interaction remain unclear. Further research integrating molecular analyses, parasite quantification, and experimental infections are necessary to clarify how parasite-parasite interactions influence sporogonic development and transmission as well as mosquito survival. These understudied aspects of malaria research are essential for improving our knowledge of avian malaria ecology of mixed parasite infections. Declarations Ethics approval The study was conducted at the State Scientific Research Institute Nature Research Centre, in the Experimental Animal Use Facility (veterinary approval number LT 61-13-003). The permit was issued by the Ethical Committee for the use of Experimental Animals at the State Food and Veterinary Service of Lithuania (Ref. No. 2015/05/27-G2-27). Consent of publication Not applicable. Competing interests The authors declare that they have no competing interests, either financial or non-financial, that are directly or indirectly related to the work submitted for publication. Funding This project has received funding from the Research Council of Lithuania (LMTLT), agreement No [S-MIP-24-85]. Author Contribution RŽ, RB, VP – experimental conception and design; RŽ – experimental infections, dissection, sporogony investigation of mosquitoes and microscopic examination; VP – experimental infections of the birds and microscopic examination; RB – molecular investigation; RB and RŽ – statistical analysis; RŽ and VP writing; all authors read and critically revised the manuscript and approved the final version of the manuscript. Acknowledgement We would like to thank Justė Aželytė for creating a figure using the “BioRender” software and sincerely thank the staff of the State Scientific Research Institute Nature Research Centre for their dedicated care of the experimental birds. Availability of data and materials The data that support the findings of this study are included within the article. 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Food availability and competition do not modulate the costs of Plasmodium infection in dominant male canaries. Exp Palasitol. 2013;135:708–14. Asghar M, Hasselquist D, Hansson B, Zehtindjiev P, Westerdahl H, Bensch S. Chronic infection. Hidden costs of infection: chronic malaria accelerates telomere degradation and senescence in wild birds. Sci. 2015;23:347(6220):436–8. Soares L, Marra P, Gray L, Ricklefs RE. The malaria parasite Plasmodium relictum in the endemic avifauna of eastern Cuba. Conserv Biol. 2017;31:1477–82. Valkiūnas G, Ilgūnas M, Bukauskaitė D, Fragner K, Weissenböck H, Atkinson CT, Iezhova TA. Characterization of Plasmodium relictum , a cosmopolitan agent of avian malaria. Malar J. 2018;17(1):184. Santiago-Alarcon D, Marzal A, editors. Avian malaria and related parasites in the tropics: ecology, evolution and systematics. Cham, Switzerland: Springer; 2020. Atkinson CT, Thomas NJ, Hunter B. Parasitic diseases of wild birds. Ames: John Wiley & Sons; 2008. Santiago-Alarcon D, Palinauskas V, Schaefer HM. Diptera vectors of avian haemosporidian parasites: untangling parasite life cycles and their taxonomy. Biol Rev. 2012;87:928–64. Palinauskas V, Žiegytė R, Iezhova TA, Ilgūnas M, Bernotienė R, Valkiūnas G. Description, molecular characterisation, diagnostics and life cycle of Plasmodium elongatum (lineage pERIRUB01), the virulent avian malaria parasite. Int J Parasitolol Parasites Wildl. 2016;46:697–707. Žiegytė R, Bernotienė R, Bukauskait D, Palinauskas, V, Iezhova T, Valkiūnas G. Complete sporogony of Plasmodium relictum (lineages pSGS1 and pGRW11) in mosquito Culex pipiens pipiens form molestus , with implications to avian malaria epidemiology. J Parasitol. 2014;100(6):878–882. Aželytė J, Wu-Chuang A, Žiegytė R, Platonova E, Mateos-Hernandez L, Maye J, Obregon D, Palinauskas V and Cabez as-Cruz A. Anti-microbiota vaccine reduces avian malaria infection within mosquito vectors. Front Immunol. 2022;13:841835. Valkiūnas G, Iezhova TA, Križanauskienė A, PalinauskasV, Bensch S. In vitro hybridization of Haemoproteus spp.: an experimental approach for direrect investigation of reproductive isolation of parasites. J Parasitol. 2008;94(6):1385–94. Valkiūnas G, Palinauskas V, Križanauskienė, Bernotienė R, Kazlauskienė R, Iezhova TA. Further observations on in vitro hybridization of hemosporidian parazites: patters of ookinete development in Haemoproteus spp. J Parasitol. 2013;99(1):124–36. Valkiūnas G, Palinauskas V, lgūnas M, Bernotienė R, Iezhova TA. In vitro development of Haemoproteus parasites: the efficiency of reproductive cells increase during simultaneous sexual process of different lineages. Parasitol Res. 2014a;113:1417–23. Marchand RP, Culleton R, Maeno Y, Quang NT, Nakazawa S. Co-infections of Plasmodium knowlesi , P. falciparum , and P. vivax among Humans and Anopheles dirus mosquitoes, southern Vietnam. Emerg Infect Dis. 2011;17(7):1232–9. Palinauskas V, Žiegytė R, Ilgūnas M, Iezhova TA, Bernotienė R, Bolshakov C, Valkiūnas G. Description of the first cryptic avian malaria parasite, Plasmodium homocircumflexum n. sp., with experimental data on its virulence and development in avian hosts and mosquitoes. Int J Parasitol. 2015;45:51–62. Godfrey RD, Fedynich AM, Pence DB. Quantification of hematozoa in blood smears. J Wildl Dis. 1987; 23:558–65. Perez-Tris J, & Bensch S. Diagnosing genetically diverse avian malarial infections using mixed-sequence analysis and TA-cloning. Parasitology. 2005;131. Kazlauskienė R, Bernotienė R, Palinauskas V, Ježova T, Valkiūnas G. Plasmodium relictum (lineages pSGS1 and pGRW11): Complete synchronous sporogony in mosquitoes Culex pipiens pipiens . Exp parasitol. 2013;133(4):454–61. Richardson DS, Jury FL, Blaakmeer K, Komdeur J, Burke T. Parentage assignment and extra-group paternity in a cooperative breeder: the Seychelles warbler ( Acrocephalus sechellensis ). Mol Ecol. 2001;10:2263–73. Bensch S, Stjenman M, Hasselquist D, Östman Ö, Hansson B, Westerdahl H, Torres-Pinheiro R. Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds. Proc R Soc B. 2000;276:1583–9. Hellgren O, Waldenström J, Bensch S A new PCR assay for simultaneous studies of Leucocytozoon , Plasmodium , and Haemoproteus from avian blood. J Parasitol. 2004;90:797–802. Valkiūnas G, Kazlauskienė R, Bernotienė,R, Palinauskas V, Ježova T. Abortive long-lasting sporogony of two Haemoproteus species (Haemosporida, Haemoproteidae) in the mosquito Ochlerotatus cantans , with perspectives on haemosporidian vector research. Parasitology research. 2013;112(6):2159–69. Valkiūnas G, Kazlauskienė R, Bernotienė R, Bukauskaitė D, Palinauskas V, Ježova T. Haemoproteus infections (Haemosporida, Haemoproteidae) kill bird-biting mosquitoes. Parasitol Res. 2014b;13(3):1011–8. Bukauskaitė D, Bernotienė R, Iezhova TA, Valkiūnas G. Mechanisms of mortality in Culicoides biting midges due to Haemoproteus infection. Parasitology. 2016;143(13):1748–54. Gutiérrez-López R, Martínez-de la Puente J, Gangoso L. Yan J, Soriguer R, Figuerola J. Experimental reduction of host Plasmodium infection load affects mosquito survival. Sci Rep. 2019;9:8782. Gupta B, Gupta P, Sharma A, Singh V, Dash AP, Das A: High proportion of mixed-species Plasmodium infections in India revealed by PCR diagnostic assay. Trop Med Int Health. 2010;15:819–24. Nakazawa S, Marchand RP, Quang NT, Culleton R, Manh ND, Maeno Y: Anopheles dirus co-infection with human and monkey malaria parasites in Vietnam. Int J Parasitol. 2009;39:1533–7. Dinko B, Oguike MC, Larbi JA, Bousema T, Sutherland CJ. Persistent detection of Plasmodium falciparum , P . malariae , P . ovale curtisi and P . ovale wallikeri after ACT treatment of asymptomatic Ghanaian school-children. Int J Parasitol Drugs Drug Resist. 2013;3:45–50. Marzal A, Bensch S, Reviriego M, Balbontin J, De Lope F. Effects of malaria double infection in birds: one plus one is not two. J Evol Biol. 2008;21(4):979–87. Ricklefs RE, Fallon, SM. Diversification and host switching in avian malaria parasites. Proc Roy Soc London Ser B Biol Sci. 2002;269:885–92. Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9368372","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626635671,"identity":"b3227e8a-7c96-439a-8fea-49df6715208a","order_by":0,"name":"Rita Žiegytė","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYDACZjApAeF8gFCMB/BrYYZpYWZgnAGkeID4AAE9CAYzDzFazNv5D35g+GMhJx+Rf/CxzS+7aHv2AwyHP+DRInOYmVmCgUfC2PBGMrNxbl9ybg9PAn5bJIB+kWCQkEjcOCOZTTq3hzm3R4KAw4BamH8wGEjUA7Ww/7bsqSdKC5sEQ4JEgrxEMhszw4/DRGkxs0g4IGG4geexsWRvw/HcnjOJDQfO4NPCf/DxjQ9/6uTl2xMffvjxpzq3vf3wwQcVeLSAQQIQG1wAkoxtIC5jAyENECDfD3L/H+IUj4JRMApGwcgCAJ7GSjhmiXg1AAAAAElFTkSuQmCC","orcid":"","institution":"State Scientific Research Institute Nature Research Centre","correspondingAuthor":true,"prefix":"","firstName":"Rita","middleName":"","lastName":"Žiegytė","suffix":""},{"id":626635672,"identity":"8999f2a7-62ac-42f2-b932-06034fe14ec2","order_by":1,"name":"Rasa Bernotienė","email":"","orcid":"","institution":"State Scientific Research Institute Nature Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Rasa","middleName":"","lastName":"Bernotienė","suffix":""},{"id":626635673,"identity":"2316aec6-a2f5-4837-bd66-d924a95bd2ab","order_by":2,"name":"Vaidas Palinauskas","email":"","orcid":"","institution":"State Scientific Research Institute Nature Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Vaidas","middleName":"","lastName":"Palinauskas","suffix":""}],"badges":[],"createdAt":"2026-04-09 12:09:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9368372/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9368372/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107488940,"identity":"ec1eb27b-ef3b-487e-a50a-072c156046ac","added_by":"auto","created_at":"2026-04-22 02:46:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":42707,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the experimental design used to assess mosquito-mediated transmission of \u003cem\u003ePlasmodium\u003c/em\u003e co-infections. Step 1: A donor canary is experimentally infected with \u003cem\u003ePlasmodium\u003c/em\u003e parasites. Step 2: Infected donor bird is exposed to \u003cem\u003eCulex p. pipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003emosquitoes to allow parasite development in the vector. Step 3: After the sporogonic development period, infected mosquitoes are allowed to bite naïve canaries to assess successful transmission and establishment of infection.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9368372/v1/68cea8ffc7a149f0af87a1f9.png"},{"id":107490408,"identity":"bc7022d3-85a1-490c-899f-2a7e44342dab","added_by":"auto","created_at":"2026-04-22 02:52:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1593359,"visible":true,"origin":"","legend":"\u003cp\u003eCo-infection of avian malaria parasites \u003cem\u003eP. relictum\u003c/em\u003e pSGS1 and \u003cem\u003eP. elongatum\u003c/em\u003e pERIRUB01 in red blood cells. (A) Gametocytes of the \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003ein different erythrocytes, (B) A single erythrocyte can contain both species of parasite when the intensity of parasitemia is high. Short arrows show \u003cem\u003eP. relictum,\u003c/em\u003e long arrows show – \u003cem\u003eP. elongatum.\u003c/em\u003e Giemsa-stained blood films. Scale bar = 10 mm.\u003c/p\u003e","description":"","filename":"Figure2scr.png","url":"https://assets-eu.researchsquare.com/files/rs-9368372/v1/37d8b384161c6cd2a26c422f.png"},{"id":109249432,"identity":"fe6e9b4a-b27d-4f9b-a181-f5711779e1bc","added_by":"auto","created_at":"2026-05-14 08:52:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2016454,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9368372/v1/d2c1985a-a17b-449e-a0fd-c9cd026e8687.pdf"},{"id":107444871,"identity":"534204d2-b33e-4e1b-984e-f5a0ef060dab","added_by":"auto","created_at":"2026-04-21 14:31:21","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":662380,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-9368372/v1/132808cdaacb283048e06765.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"The development of avian malaria co-infections in experimentally exposed vectors","fulltext":[{"header":"Background","content":"\u003cp\u003eCo-infections involving multiple haemosporidian and other parasite species are frequently observed in natural populations of humans, birds, and other vertebrates [1\u0026minus;8]. Interactions between co-infecting parasites can significantly influence disease severity, parasite development, transmission success, and host survival [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These interactions may be suppressive, facilitative, or neutral, depending on the parasite species or genetic lineages involved [9\u0026minus;12]. One key factor shaping co-infection outcomes is parasite competitiveness, which determines infection dynamics and transmission potential within hosts and vectors [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong avian malaria parasites, \u003cem\u003ePlasmodium relictum\u003c/em\u003e and \u003cem\u003ePlasmodium elongatum\u003c/em\u003e are two widespread and highly studied species. \u003cem\u003ePlasmodium relictum\u003c/em\u003e, first described by Grassi et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], is one of the most extensively investigated avian malaria parasites. Several mitochondrial DNA cytochrome \u003cem\u003eb\u003c/em\u003e (\u003cem\u003ecyt b)\u003c/em\u003e gene lineages (e.g., pSGS1, pGRW11, pGRW4, pLZFUS01, pPHCOL01) attributed to this species are deposited in GenBank and MalAvi database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mbio-serv2.mbioekol.lu.se/Malavi\u003c/span\u003e\u003cspan address=\"http://mbio-serv2.mbioekol.lu.se/Malavi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This parasite is globally distributed and recognized as an invasive pathogen responsible for severe disease outbreaks and population declines, particularly in immunologically na\u0026iuml;ve bird species [1\u0026minus;25]. The primary vectors of \u003cem\u003eP. relictum\u003c/em\u003e are mosquitoes of the genus \u003cem\u003eCulex\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003ePlasmodium elongatum\u003c/em\u003e is another pathogenic avian malaria parasite that comprises several genetic lineages, including pGRW6 and pERIRUB01. The pGRW6 lineage is particularly virulent to non-adapted bird species, such as brown kiwi and different species of penguins [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Similar to \u003cem\u003eP. relictum\u003c/em\u003e, this parasite is mainly transmitted by \u003cem\u003eCulex\u003c/em\u003e mosquitoes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Experimental studies have demonstrated that the parasite of pERIRUB01 lineage is capable of completing sporogonic development, including sporozoite formation, in \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e and \u003cem\u003eC. p. pipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003e mosquitoes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExperimental studies in avian hosts have demonstrated that outcomes of co-infections involving \u003cem\u003eP. relictum\u003c/em\u003e depend on the specific combination of parasite lineages. For example, co-infection with closely related \u003cem\u003eP. relictum\u003c/em\u003e lineages pGRW11 and pSGS1 does not alter overall parasitemia or increase virulence compared with single infections [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Experimental infections of \u003cem\u003eCulex p. pipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003e mosquitoes with these lineages showed successful sporozoite development for both parasites, although oocyst burden differed between genetic lineages [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In contrast, co-infection involving pSGS1 and \u003cem\u003eP. relictum\u003c/em\u003e lineage pGRW4 distributed in Africa resulted in transient infection in birds, with pGRW4 being suppressed and cleared from peripheral blood [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This finding suggest that locally dominant parasite lineages may competitively exclude less-adapted lineages, thereby influencing transmission success. Conversely, some interactions may be facilitative rather than competitive. For instance, the presence of the \u003cem\u003eP. relictum\u003c/em\u003e pSGS1 was shown to enhance parasitemia of the \u003cem\u003eP. elongatum\u003c/em\u003e lineage pERIRUB01 without affecting its own development, resulting in co-infection virulence similar to that of the more pathogenic parasite [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Competitive interactions have also been documented among parasites of the related genus \u003cem\u003eHaemoproteus\u003c/em\u003e in \u003cem\u003ein vitro\u003c/em\u003e experimental studies [32\u0026minus;34]. These examples demonstrate the complexity of parasite interactions during co-infection, which vary depending on parasite species, lineages, and their competitive abilities.\u003c/p\u003e \u003cp\u003eCo-infections involving \u003cem\u003ePlasmodium\u003c/em\u003e species are also common in humans and their \u003cem\u003eAnopheles\u003c/em\u003e mosquito vectors [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. For instance, sporozoites of \u003cem\u003ePlasmodium knowlesi, Plasmodium falciparum\u003c/em\u003e, and \u003cem\u003ePlasmodium vivax\u003c/em\u003e were detected in \u003cem\u003eAnopheles dirus\u003c/em\u003e mosquitoes [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, it remains unclear whether such interactions originate from a single blood meal containing multiple parasite species or from multiple feeding events. It is still uncertain whether transmission of different \u003cem\u003ePlasmodium\u003c/em\u003e species can occur through a single mosquito or require multiple infeced vectors carrying different parasite species [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, it is unclear whether competition for red blood cells occurs during co-infections, and if so, what the implications of such interaction may be McQueen and McKenzie [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. What is the effect of co-infections involving several \u003cem\u003ePlasmodium\u003c/em\u003e species on gametocyte formation? [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These questions also arise in the context of human malaria research. Despite the immense relevance of research on human malaria, experimental investigations of co-infection dynamics are limited due to ethical constraints on experimental infections in vertebrate hosts. In contrast, avian malaria system provides valuable experimental model that allow controlled investigation of parasite-parasite interactions \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAs research into avian malaria progresses, more and more information is becoming available on the interactions between parasites in co-infected vertebrate hosts. However, experimental studies of these infections in vectors are still rare despite being highly relevant [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Many investigations report only partial sporogonic stages, such as ookinetes or oocysts [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] highlighting the persistent lack of comprehensive experimental evidence on the complete development of parasites in the vectors [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on previous experimental findings and in an effort to supplement existing knowledge about parasite development in vectors we hypothesize that co-infection with \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003e in \u003cem\u003eCulex p\u003c/em\u003e. \u003cem\u003epipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003e mosquitoes will result in parallel development of both parasites to the sporozoite stage, potentially enabling their transmission during a single mosquito bite. The primary aim of this study is therefore to investigate the development, transmission potential, and virulence of \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003e during co-infection in \u003cem\u003eCulex p. pipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003e mosquitoes, and to determine whether both parasites can be simultaneously transmitted to a healthy avian host by a single vector. By examining sporogonic stages in vectors, we aim to clarify whether such co-infections require multiple vector transmission events or can occur through a single mosquito. This experimental study contributes to a broader understanding of the development, transmission strategies, and ecological implications of haemosporidian co-infections.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003ePlasmodium\u003c/b\u003e \u003cb\u003eparasites and experimental setup\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe study was performed at the State Scientific Research Institute Nature Research Centre (NRC) in Vilnius, Lithuania. Domestic canaries (\u003cem\u003eSerinus canaria domestica)\u003c/em\u003e, obtained from a commercial supplier, were used in the experiments. Prior to infection, all birds were confirmed to be uninfected with malarial parasites by both microscopic examination and PCR testing. To multiply \u003cem\u003ePlasmodium\u003c/em\u003e parasites, birds were experimentally infected by intramuscular inoculation of infected blood into the pectoral muscles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Step 1), as described by Palinauskas et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We used two avian malaria parasites \u0026ndash; \u003cem\u003eP. relictum\u003c/em\u003e pSGS1 isolated from a naturally infected \u003cem\u003eCommon crossbill\u003c/em\u003e (\u003cem\u003eLoxia curvirostra\u003c/em\u003e) and \u003cem\u003eP. elongatum\u003c/em\u003e pERIRUB01 isolated from a naturally infected \u003cem\u003eEuropean robin\u003c/em\u003e (\u003cem\u003eErithacus rubecula\u003c/em\u003e). Parasites were multiplied and subsequently cryopreserved in liquid nitrogen, following the protocol described by [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. For the infection of the donor birds we used parasites of the 3rd passage after original isolation of \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003e. We used four groups of donor birds for the experiment: birds infected with \u003cem\u003eP. relictum\u003c/em\u003e pSGS1 (group B1); birds infected with \u003cem\u003eP. elongatum\u003c/em\u003e pERIRUB1 (group B2); birds co-infected with \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003e, pSGS1\u0026times;pERIRUB1 (group B3); and uninfected control birds (group B4). Each group consisted of 6 canaries housed in individual cages in a vector-free room under controlled environmental conditions (10:14 h light/dark photoperiod, temperature 21\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg; C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003ePlasmodium relictum\u003c/em\u003e was identified following the description by Valkiūnas [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] while \u003cem\u003eP. elongatum\u003c/em\u003e was identified as described in Valkiūnas [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and Palinauskas et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For parasitological examination, approximately 100 fields were studied at high magnification (1000\u0026times;). Intensity of parasitemia was estimated as a percentage of the number of parasites per 1000 erythrocytes or per 10,000 erythrocytes if infections were light (\u0026lt;\u0026thinsp;0.1%), as recommended by Godfrey et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. We used an Olympus B51 light microscope (Olympus, Japan) with a digital camera and Olympus DP-SOFT software to analyse blood slides. The presence of a single and co-infection was confirmed by microscopic examination of blood smears and further confirmed by PCR-based methods and sequencing parasite\u0026rsquo;s DNA (see details below). Co-infections were identified by visual inspection of electropherograms based on double base-calling, as described by P\u0026egrave;rez-Tris and Bensch [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfter the development of parasitemia in the blood of infected birds, they were used as donors to infect \u003cem\u003eCulex p. pipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003e mosquitoes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Step 2) as described below. Gametocytemia was assessed in all donor canaries immediately after mosquitoes had taken a blood meal.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental infections and dissection of mosquitoes for sporogony investigation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCulex p. pipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003e mosquitoes, reared in the laboratory at the NRC, were experimentally infected with \u003cem\u003ePlasmodium\u003c/em\u003e parasites. Four groups of mosquitoes were formed based on their donor birds: mosquitoes fed on B1 group birds infected with \u003cem\u003eP. relictum\u003c/em\u003e pSGS1 (group M1); mosquitoes fed on B2 group birds infected with \u003cem\u003eP. elongatum\u003c/em\u003e pERIRUB1 (group M2); mosquitoes fed on B3 group birds co-infected with \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003e pSGS1\u0026times;pERIRUB1 (group M3); and mosquitoes fed on uninfected control birds (group M4). Mosquitoes were fed as described by Žiegytė et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and Kazlauskienė et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Briefly, infected canaries exhibiting gametocytemia were placed in a tube within an experimental mosquito cage, with only their legs exposed to mosquito bites. Blood feeding was allowed to proceed for approximately 10 minutes to 1 hour. Fully engorged female mosquitoes were taken from the cages using an aspirator, transferred to individual small insect cages, and maintained under controlled conditions (temperature 23\u0026deg; C, 60\u0026thinsp;\u0026plusmn;\u0026thinsp;2% relative humidity, and 16:8 h L/D photoperiod). All experiments were conducted in parallel.\u003c/p\u003e \u003cp\u003eDissection and preparation of permanent preparations for identifying sporogonic stages in mosquitoes (ookinetes, oocysts, and sporozoites), as well as their staining techniques, were carried out following established protocols [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Thoraxes of dissected mosquitoes were preserved in 96% ethanol for subsequent PCR-based confirmation of \u003cem\u003ePlasmodium\u003c/em\u003e lineages (see details below).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInfection of canaries by bites of experimentally infected mosquitoes\u003c/h3\u003e\n\u003cp\u003eTo evaluate the infectivity and the transmission potential of \u003cem\u003ePlasmodium\u003c/em\u003e sporozoites developed in mosquitoes with co-infections of \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003e, uninfected domestic canaries were exposed to bites of experimentally infected mosquitoes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Step 3). As recipients for the bites we used 5 healthy canaries as detected using both using microscopy and PCR. Individual mosquitoes, later confirmed to carry sporozoites in their salivary glands, were allowed to take a blood meal by placing the bird\u0026rsquo;s legs into a mesh-covered feeding cage for approximately 10\u0026minus;15 minutes as described above. Only one mosquito was allowed to feed on each bird to ensure that any subsequent infection could be traced to a single transmission event. Immediately after feeding, the mosquito was dissected, and its salivary glands were removed for microscopic preparations to determine the presence of sporozoites. Blood samples were collected from the exposed canaries at 4, 8, 12, 16, 20, and 24 days post infection (dpi) and examined by both microscopy and PCR to identify parasite species of developed blood stages.\u003c/p\u003e\n\u003ch3\u003ePCR-based examination, sequencing and statistical analysis\u003c/h3\u003e\n\u003cp\u003eTotal DNA was extracted from samples (birds\u0026rsquo; blood stored in SET buffer and mosquitoes placed in absolute ethanol) using an ammonium acetate extraction method [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. To identify parasites, we used a nested PCR protocol [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. We amplified a fragment of the \u003cem\u003ecyt b\u003c/em\u003e gene using initial primers HaemNFI and HaemNR3, which amplify DNA fragments of haemosporidians belonging to \u003cem\u003eHaemoproteus, Plasmodium\u003c/em\u003e and \u003cem\u003eLeucocytozoon\u003c/em\u003e [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and inner primers HaemF and HaemR2, which are specific to \u003cem\u003eHaemoproteus\u003c/em\u003e and \u003cem\u003ePlasmodium\u003c/em\u003e spp. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The amplification was evaluated by running 3 \u0026micro;l of the final PCR product on a 1,5% agarose gel. One negative control (nuclease-free water) and one positive control (\u003cem\u003eP. relictum\u003c/em\u003e pSGS1 microscopy positive blood) were used for every 14 samples to control for false amplifications.\u003c/p\u003e \u003cp\u003eFragments of DNA from the positive samples were sequenced using a Big Dye Terminator V3.1. Cycle Sequencing kit and an ABI PRISMTM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were analysed using BioEdit software and compared with sequences deposited in the Malavi database [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The presence of a single infection and co-infection was confirmed by visual inspection of double-base-calling at specific sequence sites on the electropherogram [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe used a chi-square (χ 2) test to assess differences between numbers of surved and dead mosquitoes.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePlasmodium\u003c/b\u003e \u003cb\u003eparasites in donor birds\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMicroscopic examination of gametocytemia in donor birds revealed intensities of 0.05% in group B1 infected with single \u003cem\u003eP. relictum\u003c/em\u003e; 0.18% in group B2 infected with single \u003cem\u003eP. elongatum\u003c/em\u003e; and 0.43% (0.09% \u003cem\u003eP. relictum\u003c/em\u003e and 0.34% \u003cem\u003eP. elongatum\u003c/em\u003e) in group B3 with co-infection. No parasites were detected in control group B4. These birds were subsequently used as donors for experimental mosquito infections. Suitable gametocytemia for healthy mosquitoes to infect birds developed approximately 8\u0026ndash;14 days after the parasites were inoculated into the birds.\u003c/p\u003e\n\u003ch3\u003eSporogony and virulence in mosquitoes\u003c/h3\u003e\n\u003cp\u003eA total of 269 \u003cem\u003eCulex p. pipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003e mosquitoes were experimentally infected: 75 mosquitoes were infected with \u003cem\u003eP. relictum\u003c/em\u003e (M1), 98 \u0026minus; with \u003cem\u003eP. elongatum\u003c/em\u003e (M2), 56 \u0026minus; with co-infection (M3), and 40 mosquitoes served as uninfected controls (M4) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe virulence of \u003cem\u003ePlasmodium\u003c/em\u003e infections in \u003cem\u003eCulex p. pipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003e mosquitoes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of infected mosquitoes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of dead mosquitoes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMortality\u003c/p\u003e \u003cp\u003erate (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM1 (\u003cem\u003eP. relictum\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM2 (\u003cem\u003eP. elongatum\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e63.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM3 (co-infection)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM4 (uninfected mosquitoes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSporogonic development in M1 group was completed in 37 (90.2%) of 41 surviving mosquitoes between 20 and 23 days post exposure (dpe) with sporozoites observed in the salivary glands. For group M2, sporogony was completed in 14 (38.9%) of 36 surviving mosquitoes between 20 and 25 dpe. In the co-infection group M3, 15 (40.5%) of 37 surviving mosquitoes completed sporogony for both parasites within the same 20\u0026ndash;25 dpe period. The presence of single and co-infection of \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003e in salivary glands was confirmed using PCR and sequencing by visual inspection of double-base-calling in specific locuses in sequence electropherograms. Even though the sporozoite timing was similar across all experimental groups, the mortality rates of mosquitoes varied. The development of a single \u003cem\u003eP. relictum\u003c/em\u003e (M1) did not result in a higher mortality rate among the exposed insects (34 out of 75, or 45.3%) compared with the control group (40.0%) until 20 dpe (p\u0026thinsp;=\u0026thinsp;0.6) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, the mortality of mosquitoes infected with \u003cem\u003eP. elongatum\u003c/em\u003e (M2) was significantly higher, reaching 63.3%, with a significant difference in mortality between this group and the group M1 (p\u0026thinsp;=\u0026thinsp;0.02, χ\u0026sup2; = 5.53) as well as the M3 group, infected with both parasites (p\u0026thinsp;=\u0026thinsp;0.0005, χ\u0026sup2; = 12.3) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Mortality of the M2 group did not differ significantly from that of the control group M4 (p\u0026thinsp;=\u0026thinsp;0.4). Similarly, mortality of the M3 group did not differ from that of the control group M4 (p\u0026thinsp;=\u0026thinsp;0.7).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransmission of\u003c/b\u003e \u003cb\u003ePlasmodium\u003c/b\u003e \u003cb\u003eparasites to birds via a single mosquito bite\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the infectivity of co-infection-derived sporozoites, mosquitoes with co-infection (M3) were allowed to take a blood meal from healthy canaries. Successful development of single infections of investigated parasites in \u003cem\u003eCulex p. pipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003e mosquitoes fed on experimentally infected canaries was proved in our previous studies [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], so in order to reduce the number of animals used for experimental procedures, we did the experiment of the final transmission only with co-infected mosquitoes. The co-infected mosquitoes from group M3 were allowed to feed on the blood of 5 healthy canaries, with a single mosquito feeding on the blood of a single canary. The mosquitoes were immediately dissected after the blood meal, and sporozoites were detected in their salivary glands. In two of the five canaries, parasites of both \u003cem\u003ePlasmodium\u003c/em\u003e species developed, and the birds became co-infected, as determined by microscopy and PCR-based methods (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Sequencing confirmed the presence of both \u003cem\u003eP. relictum\u003c/em\u003e pSGS1 and \u003cem\u003eP. elongatum\u003c/em\u003e pERIRUB1 lineages in two experimentally infected birds, as evidenced by double base calls in DNA sequencing chromatograms at diagnostic nucleotide positions where the two sequences differ. In one double infected canary, only \u003cem\u003eP. relictum\u003c/em\u003e parasite successfully developed, while the other two canaries remained uninfected, despite the detection of parasite sporozoites in mosquitoes that had fed on them. Between 8 and 16 dpi, parasites of both \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003e were identified in blood smears from co-infected birds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides novel insights into the development and transmission potential of two distantly related avian malaria parasites, \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003e, during co-infection in \u003cem\u003eC. p. pipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003e mosquitoes. Our results reveal that both parasite species can complete sporogonic development within the same vector and can be simultaneously transmitted to a na\u0026iuml;ve avian host through a single mosquito bite. Additionally, the study shows distinct effects of single and co-infections on mosquito survival, suggesting that parasite interactions during sporogony may influence vector fitness.\u003c/p\u003e \u003cp\u003eSporozoites of both \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003e were detected in the salivary glands of infected mosquitoes between 15 and 21 dpe. These results are consistent with previous studies showing that \u003cem\u003eP. relictum\u003c/em\u003e completes sporogonic development in \u003cem\u003eCulex p. pipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003e mosquitoes within approximately 14\u0026ndash;32 dpe, with sporozoites commonly observed in exposed insects [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Although \u003cem\u003eP. relictum\u003c/em\u003e is capable of completing sporogony in more than 25 mosquito species belonging to different genera, mosquitoes of the genus \u003cem\u003eCulex\u003c/em\u003e are considered its primary vectors [19,26\u0026minus;28]. For \u003cem\u003eP. elongatum\u003c/em\u003e (pERIRUB01), sporozoites were observed between 18 and 23 dpe in this study, which is comparable to earlier experimental findings reporting sporozoite formation between 20 and 22 dpe in \u003cem\u003eCulex p. pipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003e, where sporozoites were detected in approximately 13\u0026ndash;20% of exposed mosquitoes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Historically, attempts to identify \u003cem\u003eP. elongatum\u003c/em\u003e vectors involved several mosquito genera, including \u003cem\u003eAedes, Anopheles, Culex\u003c/em\u003e, and \u003cem\u003eCuliseta\u003c/em\u003e, but many of these early studies produced inconclusive results [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe observed differences in mosquito mortality among infection groups demonstrate substantial variation in vector tolerance depending on infecting \u003cem\u003ePlasmodium\u003c/em\u003e species. Mosquitoes infected with \u003cem\u003eP. relictum\u003c/em\u003e alone did not exhibit elevated mortality compared with uninfected controls. This observation is consistent with previous studies suggesting that the pSGS1 lineage of \u003cem\u003eP. relictum\u003c/em\u003e may cause relatively low virulence in some vertebrate hosts [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In contrast, mosquitoes infected with \u003cem\u003eP. elongatum\u003c/em\u003e alone experienced significantly higher mortality, a pattern that aligns with the high pathogenicity of this parasite in avian host [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Interestingly, mosquitoes co-infected with both parasites showed lower mortality than those infected with \u003cem\u003eP. elongatum\u003c/em\u003e alone. This pattern may indicate a suppressive interaction, whereby \u003cem\u003eP. relictum\u003c/em\u003e partially limits the development of \u003cem\u003eP. elongatum\u003c/em\u003e within the mosquito. Such suppression could reduce physiological stress on the vector and thereby improve survival. However, further targeted experiments are necessary to determine whether this effect reflects direct parasite competition, immune-mediated interactions, or resource limitation within the mosquito. Vector mortality is sometimes associated with the intensity of parasitaemia, i.e. parasite burden in the blood of infected birds, as demonstrated for \u003cem\u003eHaemoproteus\u003c/em\u003e parasites [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In the present study, however, mosquito mortality did not correlate with parasite gametocytemia in donor birds. The highest gametocitemia occurred in birds with co-infection, reaching 0.43%, and mosquitoes in this group showed the lowest mortality (33.9%), comparable to the control group. In contrast, the highest mortality (63.2%) was observed in the group infected with single \u003cem\u003eP. elongatum\u003c/em\u003e, where the donor bird exhibited only 0.18% gametocytemia. Previous studies suggest that high parasite burdens may reduce mosquito survival, potentially making parasite transmission more efficient during the chronic rather than acute infection stage [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is notable that co-infections involving \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003e are known to be highly virulent and often lethal in birds [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], whereas in mosquitoes the same parasite combination appears less harmful than infection with single \u003cem\u003eP. elongatum\u003c/em\u003e. This contrast highlights the complexity of host-parasite-vector interactions and indicates that the fitness costs of co-infections may differ substantially between vertebrate and invertebrate hosts.\u003c/p\u003e \u003cp\u003eComplex interactions between co-infecting haemosporidian parasites have also been demonstrated in experimental studies of \u003cem\u003eHaemoproteus\u003c/em\u003e parasites. \u003cem\u003eIn vitro\u003c/em\u003e experiments have shown complete inhibition of ookinete development between certain parasite species, such as \u003cem\u003eHaemoproteus pallidus\u003c/em\u003e and \u003cem\u003eHaemoproteus minutus\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and between \u003cem\u003eH. pallidus\u003c/em\u003e and \u003cem\u003eHaemoproteus tartakovskyi\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Conversely, the experiments with \u003cem\u003eH. tartakovskyi\u003c/em\u003e and \u003cem\u003eHaemoproteus parabelopolskyi\u003c/em\u003e have demonstrated increased reproductive activity and a higher number of normaly forming ookinetes of the first parasite, but the blockage of ookinete development of \u003cem\u003eH. tartakovskyi\u003c/em\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These examples show that parasite-parasite interactions during sexual reproduction can range from strong competition to facilitation and may substantially influence transmission success.\u003c/p\u003e \u003cp\u003eThe most significant finding of this study is the successful transmission of two genetically distant \u003cem\u003ePlasmodium\u003c/em\u003e species from a single co-infected mosquito to a na\u0026iuml;ve canary host during a single feeding event. To our knowledge, this is the first experimental demonstration of simultaneous transmission of two avian malaria parasites by a single mosquito bite. Historical literature shows that co-infections of \u003cem\u003ePlasmodium\u003c/em\u003e species are common in human malaria systems [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. For example, infections with \u003cem\u003eP. vivax\u003c/em\u003e frequently occur following \u003cem\u003eP. falciparum\u003c/em\u003e malaria episodes in approximately one-third of cases [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, available entomological evidence suggests that most of these co-infections likely result from sequential inoculation events by different mosquitoes rather than from a single mosquito bite [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].Our experimental results indicate that simultaneous transmission by a single vector is nevertheless possible. Similar patterns may occur in other malaria systems. Holzschuh et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] reported that \u003cem\u003ePlasmodium malariae\u003c/em\u003e is detected more frequently in individuals co-infected with \u003cem\u003eP. falciparum\u003c/em\u003e \u0026minus; of the four parasite species \u003cem\u003eP\u003c/em\u003e. \u003cem\u003evivax\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e. \u003cem\u003emalariae\u003c/em\u003e, and \u003cem\u003eP\u003c/em\u003e. \u003cem\u003eovale\u003c/em\u003e, that were circulating simultaneously, \u003cem\u003eP. malaria\u003c/em\u003e benefited the most from being co-infected with the others, suggesting that interactions between parasite species may facilitate the persistence or spread of certain parasites. Obtained gametocytes of both species with a single blood meal should result, at least in some cases, in the transmission of both parasite species during a single mosquito bite. Such assumptions could be made based on our experimental data; however, there is a possibility that these specific parasites may inhibit sporogonic development in the vector, even if they both can persist simultaneously in the blood stream of the vetebare host. Similarly, depending on their relative reproduction rates and the timing of inoculation, one species may suppress the other when competing for red blood cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Although co-infections are widely documented in wild bird and human populations [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], they are generally assumed to result from sequential bites by multiple infected mosquitoes or from long-term parasite persistence within the host. The demonstration that \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003e can be transmitted simultaneously by a single mosquito challenges this assumption and suggests that single vector-mediated transmission may contribute more strongly to mixed infecions than previously recognized.\u003c/p\u003e \u003cp\u003eThe capacity of a single mosquito to transmit multiple parasite species may influence within-host parasite competition, host immune responses, interactions with the host microbiome, and the structure of parasite communities. Such transmission events may facilitate the establishment of mixed infections and potentially alter disease outcomes in avian hosts [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, vectors harboring multiple \u003cem\u003ePlasmodium\u003c/em\u003e lineages may serve as environments where genetic recombination can occur, potentially increasing parasite genetic diversity and adaptive potential [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnderstanding the dynamics of parasite interactions within vectors is an understudied aspect of malaria research. Molecular approaches such as quantitative PCR (\u003cem\u003eq\u003c/em\u003ePCR) could help address this gap by enabling precise quantification of each parasite species abundance during sporogonic development. Applying such technique to various combinations of \u003cem\u003ePlasmodium\u003c/em\u003e species would substantially advance our understanding of parasite competition in mosquitoes and transmission dynamics.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates that co-infection with \u003cem\u003eP. relictum\u003c/em\u003e and \u003cem\u003eP. elongatum\u003c/em\u003e can successfully complete sporogonic development to infective sporozoite stages in \u003cem\u003eC. p. pipiens\u003c/em\u003e f. \u003cem\u003emolestus\u003c/em\u003e mosquitoes. Sporozoites of both parasite species were detected in mosquito salivary grands between 21 and 23 dpe and both parasites were simultaneously transmitted to a na\u0026iuml;ve avian host through a single mosquito bite. Mosquitoes infected with both parasite species exhibited lower mortality than those infected with \u003cem\u003eP. elongatum\u003c/em\u003e alone, suggesting a potential suppressive interaction between parasites within the vector. However, the mechanisms underlying this interaction remain unclear. Further research integrating molecular analyses, parasite quantification, and experimental infections are necessary to clarify how parasite-parasite interactions influence sporogonic development and transmission as well as mosquito survival. These understudied aspects of malaria research are essential for improving our knowledge of avian malaria ecology of mixed parasite infections.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003e The study was conducted at the State Scientific Research Institute Nature Research Centre, in the Experimental Animal Use Facility (veterinary approval number LT 61-13-003). The permit was issued by the Ethical Committee for the use of Experimental Animals at the State Food and Veterinary Service of Lithuania (Ref. No. 2015/05/27-G2-27).\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent of publication\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests, either financial or non-financial, that are directly or indirectly related to the work submitted for publication.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis project has received funding from the Research Council of Lithuania (LMTLT), agreement No [S-MIP-24-85].\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRŽ, RB, VP \u0026ndash; experimental conception and design; RŽ \u0026ndash; experimental infections, dissection, sporogony investigation of mosquitoes and microscopic examination; VP \u0026ndash; experimental infections of the birds and microscopic examination; RB \u0026ndash; molecular investigation; RB and RŽ \u0026ndash; statistical analysis; RŽ and VP writing; all authors read and critically revised the manuscript and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003e We would like to thank Justė Aželytė for creating a figure using the \u0026ldquo;BioRender\u0026rdquo; software and sincerely thank the staff of the State Scientific Research Institute Nature Research Centre for their dedicated care of the experimental birds.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are included within the article. The preparations of vector stages were deposited in the State Scientific Research Institute Nature Research Centre, Vilnius, Lithuania.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGarnham PCC. Malaria parasites and other haemosporidia. Oxford: Blackwell; 1966.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCox FEG. Concomitant infections, parasites and immune responses. Parasitology. 2001;122:23\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValkiūnas G, Bensch S, Iezhova TA, Krizanauskien\u0026eacute; A, Hellgren O, Bolshakov CV. Nested cytochrome \u003cem\u003eb\u003c/em\u003e polymerase chain reaction diagnostics underestimate mixed infections of avian blood haemosporidian parasites: microscopy is still essential. J Parasitol. 2006;92(2):418\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eImwong M, Nakeesathit S, Day NPJ, White NJ. 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Further observations on \u003cem\u003ein vitro\u003c/em\u003e hybridization of hemosporidian parazites: patters of ookinete development in \u003cem\u003eHaemoproteus\u003c/em\u003e spp. J Parasitol. 2013;99(1):124\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValkiūnas G, Palinauskas V, lgūnas M, Bernotienė R, Iezhova TA. \u003cem\u003eIn vitro\u003c/em\u003e development of \u003cem\u003eHaemoproteus\u003c/em\u003e parasites: the efficiency of reproductive cells increase during simultaneous sexual process of different lineages. Parasitol Res. 2014a;113:1417\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarchand RP, Culleton R, Maeno Y, Quang NT, Nakazawa S. Co-infections of \u003cem\u003ePlasmodium knowlesi\u003c/em\u003e, \u003cem\u003eP. falciparum\u003c/em\u003e, and \u003cem\u003eP. vivax\u003c/em\u003e among Humans and \u003cem\u003eAnopheles dirus\u003c/em\u003e mosquitoes, southern Vietnam. Emerg Infect Dis. 2011;17(7):1232\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalinauskas V, Žiegytė R, Ilgūnas M, Iezhova TA, Bernotienė R, Bolshakov C, Valkiūnas G. Description of the first cryptic avian malaria parasite, \u003cem\u003ePlasmodium homocircumflexum\u003c/em\u003e n. sp., with experimental data on its virulence and development in avian hosts and mosquitoes. Int J Parasitol. 2015;45:51\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGodfrey RD, Fedynich AM, Pence DB. Quantification of hematozoa in blood smears. J Wildl Dis. 1987; 23:558\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez-Tris J, \u0026amp; Bensch S. Diagnosing genetically diverse avian malarial infections using mixed-sequence analysis and TA-cloning. Parasitology. 2005;131.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKazlauskienė R, Bernotienė R, Palinauskas V, Ježova T, Valkiūnas G. \u003cem\u003ePlasmodium relictum\u003c/em\u003e (lineages pSGS1 and pGRW11): Complete synchronous sporogony in mosquitoes \u003cem\u003eCulex pipiens pipiens\u003c/em\u003e. Exp parasitol. 2013;133(4):454\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichardson DS, Jury FL, Blaakmeer K, Komdeur J, Burke T. Parentage assignment and extra-group paternity in a cooperative breeder: the Seychelles warbler (\u003cem\u003eAcrocephalus sechellensis\u003c/em\u003e). Mol Ecol. 2001;10:2263\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBensch S, Stjenman M, Hasselquist D, \u0026Ouml;stman \u0026Ouml;, Hansson B, Westerdahl H, Torres-Pinheiro R. Host specificity in avian blood parasites: a study of \u003cem\u003ePlasmodium\u003c/em\u003e and \u003cem\u003eHaemoproteus\u003c/em\u003e mitochondrial DNA amplified from birds. Proc R Soc B. 2000;276:1583\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHellgren O, Waldenstr\u0026ouml;m J, Bensch S A new PCR assay for simultaneous studies of \u003cem\u003eLeucocytozoon\u003c/em\u003e, \u003cem\u003ePlasmodium\u003c/em\u003e, and \u003cem\u003eHaemoproteus\u003c/em\u003e from avian blood. J Parasitol. 2004;90:797\u0026ndash;802.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValkiūnas G, Kazlauskienė R, Bernotienė,R, Palinauskas V, Ježova T. Abortive long-lasting sporogony of two \u003cem\u003eHaemoproteus\u003c/em\u003e species (Haemosporida, Haemoproteidae) in the mosquito \u003cem\u003eOchlerotatus cantans\u003c/em\u003e, with perspectives on haemosporidian vector research. 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Sci Rep. 2019;9:8782.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta B, Gupta P, Sharma A, Singh V, Dash AP, Das A: High proportion of mixed-species \u003cem\u003ePlasmodium\u003c/em\u003e infections in India revealed by PCR diagnostic assay. Trop Med Int Health. 2010;15:819\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakazawa S, Marchand RP, Quang NT, Culleton R, Manh ND, Maeno Y: Anopheles dirus co-infection with human and monkey malaria parasites in Vietnam. Int J Parasitol. 2009;39:1533\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDinko B, Oguike MC, Larbi JA, Bousema T, Sutherland CJ. Persistent detection of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e. \u003cem\u003emalariae\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e. \u003cem\u003eovale curtisi\u003c/em\u003e and \u003cem\u003eP\u003c/em\u003e. \u003cem\u003eovale wallikeri\u003c/em\u003e after ACT treatment of asymptomatic Ghanaian school-children. Int J Parasitol Drugs Drug Resist. 2013;3:45\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarzal A, Bensch S, Reviriego M, Balbontin J, De Lope F. Effects of malaria double infection in birds: one plus one is not two. J Evol Biol. 2008;21(4):979\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRicklefs RE, Fallon, SM. Diversification and host switching in avian malaria parasites. Proc Roy Soc London Ser B Biol Sci. 2002;269:885\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Culex pipiens, co-infections, mosquitoes, Plasmodium relictum, Plasmodium elongatum, sporozoites","lastPublishedDoi":"10.21203/rs.3.rs-9368372/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9368372/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCo-infections with multiple parasites are widespread in wild populations and can influence both host-parasite and vector-parasite interactions. Among haemosporidian parasites, co-infections involving different genera or occasionally, multiple species within the same genus \u0026ndash; such as \u003cem\u003ePlasmodium\u003c/em\u003e \u0026ndash; have been documented in birds. However, the impact of such co-infections on sporogonic development within vectors remains poorly understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study investigated the sporogony, transmission and virulence of single and co-infections of \u003cem\u003ePlasmodium relictum\u003c/em\u003e (genetic lineage pSGS1) and \u003cem\u003ePlasmodium elongatum\u003c/em\u003e (genetic lineage pERIRUB1) in \u003cem\u003eCulex pipiens pipiens\u003c/em\u003e form \u003cem\u003emolestus\u003c/em\u003e mosquitoes, using microscopic examination and PCR testing. Mosquitoes were experimentally infected by allowing them to take a blood meal from canaries carrying either a parasite of a single species or exposed to birds infected with two species, resulting in double infection. After blood meals, the insects were maintained until the parasite reached the sporozoite stage.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur findings reveal that both parasite species can complete sporogonic development within the same insect vector and can be simultaneously transmitted to a healthy avian host through a single mosquito bite. Additionally, the study shows distinct effects of single and co-infections on mosquito survival, demonstrating specific interactions between parasites during sporogonic development in the vector. Co-infected mosquitoes experienced lower mortality than those infected with a single \u003cem\u003eP. elongatum\u003c/em\u003e infection. This could be due to a potential suppressive effect of \u003cem\u003eP. relictum\u003c/em\u003e on the development of \u003cem\u003eP. elongatum\u003c/em\u003e within the mosquito, thereby reducing harm to the vector.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study deepens our understanding of avian malaria co-infections by focusing on co-infections within vectors, and highlights their potential impact on transmission efficiency, vector fitness and the ecological dynamics of disease spread.\u003c/p\u003e","manuscriptTitle":"The development of avian malaria co-infections in experimentally exposed vectors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-21 14:31:16","doi":"10.21203/rs.3.rs-9368372/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b9f5bb7a-58c6-4b35-801f-0e6d9a659b42","owner":[],"postedDate":"April 21st, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-09T18:32:04+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-09T18:39:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-21 14:31:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9368372","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9368372","identity":"rs-9368372","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}