Currently, there are very few studies of avian malaria that investigate relationships among the host-vector-parasite triad concom- itantly. In the current study, we experimentally measured the vector competence of several Culex mosquitoes for a newly described avian malaria parasite, Plasmodium homopolare. Song sparrow (Melospiza melodia) blood infected with a low P. homopolare parasitemia was inoculated into a naïve domestic canary (Serinus canaria forma domestica). Within5to10dayspostinfection (dpi), the canary unexpectedly developed a simultaneous high parasitemic infection of Plasmodium cathemerium (Pcat6) and a low parasitemic infection of P. homopolare, both of which were detected in blood smears. During this infection period, PCR detected Pcat6, but not P. homopolare in the canary. Between 10 and 60 dpi, Pcat6 blood stages were no longer visible and PCR no longer amplified Pcat6 parasite DNA from canary blood. However, P. homopolare blood stages remained visible, albeit still at very low parasitemias, and PCR was able to amplify P. homopolare DNA. This pattern of mixed Pcat6 and P. homopolare infection was repeated in three secondary infected canaries that were injected with blood from the first infected canary. Mosquitoes that blood-fed on the secondary infected canaries developed infections with Pcat6 as well as another P. cathemerium lineage (Pcat8); none developed PCR detectable P. homopolare infections. These observations suggest that the original P. homopolare-infected songbird also had two un-detectable P. cathemerium lineages/strains. The vector and host infectivity trials in this study demonstrated that current molecular assays may significantly underreport the extent of mixed avian malaria infections in vectors and hosts. . . . . . Keywords Avian malaria Experimental infection Co-infection Plasmodium cathemerium Plasmodium homopolare Culex mosquitoes Introduction determining the transmission rates of pathogens in a communi- ty (McCallum et al. 2001). The task of separating minor from In vector-borne disease systems, identifying the relative contri- major vectors is relatively easily accomplished in simple avian bution of different vector and host species is a crucial step in malaria systems such as in the Hawaiian archipelago, where * Jenny S. Carlson Department of Entomology, University of California at Davis, email@example.com Davis, CA, USA Lake County Vector Control, Lakeport, CA, USA Brittany Nelms firstname.lastname@example.org Department of Pathology, Microbiology, and Immunology, Christopher M. Barker University of California at Davis, Davis, CA, USA email@example.com Department of Biology, San Francisco State University, San William K. Reisen Francisco, CA, USA firstname.lastname@example.org Vector Genetics Laboratory, Dept. Pathology, Microbiology and Ravinder N. M. Sehgal Immunology, University of California at Davis, Davis, CA, USA email@example.com Anthony J. Cornel Adjunct Appointment, School of Health Systems & Public Health, firstname.lastname@example.org University of Pretoria, Pretoria, South Africa 2386 Parasitol Res (2018) 117:2385–2394 one Plasmodium and few mosquito species co-exist (Van Riper Materials and methods et al. 1986; LaPointe et al. 2012; Winchester and Kapan 2013). Far more complex vector-vertebrate and parasite-host-vector Infected wild bird blood collection interactions occur in systems of multiple host and vector spe- cies (Dietz 1980). In most locations, several vectors contribute Infected blood from one song sparrow (Melospiza melodia) to the transmission of multiple avian pathogens, and in the was obtained from China Creek Park, Central California, context of disease dynamics, the general dimension of vector following methods described by Walther et al. (2016). functional diversity is an important consideration. However, Following protocols designed by Carlson et al. (2016), Power and Flecker (2008) state that functional diversity in- 100 μl of song sparrow blood was extracted by jugular cludes many factors not necessarily related just to vector taxo- venipuncture with a syringe preloaded with 0.014 cc of nomic diversity. Compatibility with both the host and the vec- citrate phosphate dextrose adenine (CPDA) solution to tor, along with abiotic factors (such as environmental con- prevent clotting. Half of the blood drawn into the syringe straints and temperature), will determine the biogeographical was discharged into a tube containing lysis buffer (10 mM distribution of parasites and is a product of co-evolution be- Tris-HCL, pH 8.0, 100 mM EDTA, 2% SDS) and held at tween parasites, hosts, and vectors (Kawecki 1998). room temperature for later DNA extraction and molecular Additionally, all parameters of the vectorial capacity of one testing. The syringe holding the remainder of the blood vector species may vary considerably in time and space due was placed in a plastic bag and held on top of wet ice to genetic polymorphisms in different populations and ecosys- for transport to the laboratory (for 174 miles). Two thin tems (Lambrechts et al. 2009). blood smear slides air-dried and fixed in absolute metha- We attempted to investigate avian malaria dynamics in cen- nol in the field were also made from small aliquots of the tral California, where large numbers of resident and migratory song sparrow blood. The slides were stained, the same day songbirds and multiple Culex, Culiseta,and Aedes mosquito the smear was made, with Giemsa as described by species occur. Prevalence studies conducted in 2011 and 2012 Valkiūnas et al. (2008). The intensity of parasitemia, esti- showed that China Creek Park harbored a rich diversity of mated by counting the number of parasites per 10,000 avian Plasmodium species and lineages in birds (Walther et erythrocytes, was determined to be 0.0002%. Parasites al. 2016) and mosquitoes (Carlson et al. 2015). However, there were identified both by microscopy using morphological was some incongruence between the prevalence of keys described by Valkiūnas (2005) and by sequencing Plasmodium species in resident birds and mosquitoes. Some parasite DNA after amplification via PCR. Parasite DNA parasite species identified in resident birds were not found in was extracted from the whole blood sample following the the local mosquitoes and vice versa. One parasite species, DNeasy blood and tissue kit protocols (Qiagen, Valencia, Plasmodium homopolare (belonging to the subgenus CA). A nested PCR described in Waldenström et al. Novyella), newly described by Walther et al. (2014), was the (2004) was carried out to amplify a 478-bp sequence of most common parasite in resident birds but was rarely found in the mitochondrial cytochrome b gene (cyt b). All PCR mosquitoes (Walther et al. 2014; Carlson et al. 2015). This products were viewed on 1.8% agarose gels stained with anomaly led us to hypothesize that some ornithophilic Culex ethidium bromide. The positive sample was cleaned and sent mosquito species were incompatible vectors of some for sequencing to Elim Biopharmaceuticals Inc. (Hayward, Plasmodium parasite species. To test this hypothesis, we CA). The sequences were edited using Sequencher 5.1 attempted to infect Culex mosquito species with P. homopolare. (Gene Codes, Ann Arbor, MI) and were then identified using Because we were unable to culture P. homopolare, we collected aNCBI nucleotide BLAST search. this parasite from a wild bird that was identified as positive for P. homopolare by microscopy and polymerase chain reaction Inoculation of donor blood into canaries (PCR). In a controlled laboratory setting, this blood extract was injected into a domestic canary (Serinus canaria forma Microscopic examinations and PCR screening of the pe- domestica). Blood from this canary was subsequently injected ripheral blood of all four domestic canaries (approximately into three other canaries, which were in turn used to infect 2–3 years of age), obtained from a California breeder mosquitoes to determine vector competence. (Steve Mieser, as described in our IACUC permit 17601) In nature, multiple instances and opportunities occur within and used for the infectivity studies, revealed that they were the vertebrate and invertebrate hosts for complex parasite- not infected with malaria parasites prior to the experimen- parasite interactions that ultimately impact the heterogeneity, tation. We confirmed with the breeder that the canaries persistence, and transmission dynamics of Plasmodium in a were not exposed to mosquitoes because they were main- community. Here, we describe the results from the vector- tained in an indoor aviary. The canaries were tested upon competence studies and discuss how these data align with arrival and again 15 and 30 days after arrival via blood the prevalence data collected in the field at China Creek Park. smear inspection and PCR analysis of collected blood Parasitol Res (2018) 117:2385–2394 2387 samples. One of the canaries (canary A) was injected with Experimental infection of mosquitoes 0.04 cc of infected song sparrow whole blood into the jugular vein. Ten days post infection (dpi), a blood smear All adult mosquitoes were maintained in an incubator at 26 °C was made with whole blood of canary A extracted from the and 70% humidity with an automatic 12 h light/dark cycle. Up brachial vein to check for the establishment of an infection. to 50 mosquitoes were placed in each one-gallon bucket and Infected blood from canary A was injected into canaries B were provided with four cotton balls lightly soaked in 10% and C as a second passage. One last direct canary-to- sucrose that were replaced daily. canary inoculation was conducted from canary C to canary To increase the probability of a successful blood feeding, D (a third passage)—refer to Fig. 1 for the schematic. all mosquitoes were starved by removing their sugar access 24 h prior to blood feeding on the infected canaries. Mosquitoes were aspirated from their original cage to a 3.7-l Collection and rearing of field mosquitoes bucket that contained the unrestrained infected canary. The canaries were unrestrained to reduce stress on the bird that Adult female mosquitoes used in the experimental infections was infected with malaria and allow for a more natural included Culex quinquefasciatus (colony mosquitoes obtained feeding. The mosquitoes were allowed to feed for 1 h on from the Greater Los Angeles County Vector Control District), each canary starting from 20 to 2100 h to emulate the Cx. pipiens complex (collected at China Creek Park, Fresno crepuscular feeding pattern. The feeding was supervised County, 36° 43′ 27.0″ N, 119° 30′ 07.3″ W), and Cx. for the entire hour to ensure that no more than ~ 50 mos- stigmatosoma (collected by the Lake County Vector Control quitoes (half of the mosquitoes in the cage) fed on one District at the Steele Winery, 39° 59′ 35.999″ N, 122° 52′ 31″ canary at a time. We determined that up to 100 mosquitoes W). Wild Cx. tarsalis were originally collected at the Yolo could safely feed on a canary at a time since each mosquito Bypass Wildlife Area (38° 33′ 53.01″ N, 121° 35′ 41.621″ W), can take a maximum of 3 μl of blood per feeding (Klowden but because they all died within a few days after imbibing blood and Lea 1978). A canary weighs on average 30 g so if 100 from the canaries, colony mosquitoes for this species were ob- mosquitoes take 3 μl of blood, then a maximum of 300 μl tained at the Sutter-Yuba Mosquito and Vector Control District. will be taken in total for each feeding, which is 1% of the F1 adults were reared from egg rafts collected from the surface body weight of a bird. However, a maximum cut-off was of ponds and also from eggs laid from gravid females, which not necessary because it was rare that more than 15 mosquitoes were originally collected in gravid traps (Cummings 1992; at a time would feed on the same canary. Reiter 1987). Cx. pipiens complex member species were identi- Fully blood-fed mosquitoes were transferred into smaller fied by PCR, following the protocol described by Smith and half-liter cartons (no more than 25 individuals per carton) and Fonseca (2004), as Cx. pipiens, Cx. quinquefasciatus, and hy- held in the incubator. Mosquitoes that did not feed were placed brids of the two species. However, we use caution with these back into their original 3.7-l container and were used again in identifications for Kothera et al. (2013) were not able to find subsequent feeding attempts, while partially blooded mosqui- pure Cx. pipiens in California, only hybrids. toes were discarded. Fully blood-fed mosquitoes were Fig. 1 Schematic of Plasmodium homopolare passage from a field- caught song sparrow (Melospiza melodia) to naïve domestic ca- naries (Serinus canaria forma domestica). A mosquito symbol indicates the canaries upon which experimental mosquitoes took an infectious blood meal (see Table 1 for more details) 2388 Parasitol Res (2018) 117:2385–2394 monitored daily, and any individuals that died prior to or be- Data analysis tween scheduled time points for dissections (15, 20, and 25 dpi) were placed into 70% ethanol until processed with the Salivary gland infection rates, determined by PCR as de- experimental mosquito samples. scribed in Carlson et al. (2015), were tested for variation be- tween mosquito species using a logistic regression analysis. Determination of infection in experimental The analysis modeled the proportion of positive salivary mosquitoes glands against the day of infection and tested whether the overall proportion of mosquitoes with sporozoites in their sal- To determine the infectious status, some females of each mos- ivary glands differed between mosquito species after adjust- quito species were dissected on 15, 20, and 25 days post ment for any time trend. All analyses were performed in R infection (dpi). A period of 15 days at 26 °C was considered version 3.2 (R Core Team 2015). long enough for sporozoites to have migrated to the salivary glands based on vector competence trials of other Plasmodium species (Meyer and Bennet 1976; Kazlauskiené et al. 2013; Carlson et al. 2016). The number of mosquitoes dissected Results depended on the availability of mosquitoes of each species still surviving after 15 to 25 dpi. All mosquitoes were first Canary infections anesthetized with triethylamine (Sigma-Aldrich, St. Louis, MO). Salivary glands were dissected from the thorax and the Canary Awas believed to be infected with only P. homopolare midgut from the abdomen, and slide preparations made. (lineage SOSP_CA3P; GenBank accession number Thoraxes and salivary glands were placed in separate tubes KJ482708); however, recipient canary A was subsequently containing 70% ethanol and were used for parasite DNA ex- found to be co-infected with both P. homopolare and P. traction and amplification in the same manner described by cathemerium. P. cathemerium lineage SPTO_CA_ELW_6P Carlson et al. (2015). Preparations of midguts to detect oo- (Pcat6; GenBank accession number KJ620779) infection with cysts were made by removing the midgut and placing it into a 3.6% merozoites was first detected on 10 dpi in canary A, and drop of saline, followed by adding a drop of 0.5% solution of gametocytes were detected on 5 dpi in the two secondary mercurochrome. Because no permanent preparations of mid- infected canaries (B and C). P. cathemerium parasitemias in guts were made, each midgut was viewed microscopically canaries B and C ranged from 5.5 to 6.7%, respectively. within 20 min of preparation. Canary D, which was infected with blood from canary C, The ideal way to determine vector competence is to have had the highest P. cathemerium parasitemia on 5 dpi at 37%. infected vectors refeed on a naïve host. We attempted In all canaries, P. cathemerium parasitemias declined after 5 refeeding mosquitoes on two Plasmodium-negative canaries, dpi to < 1% on 7 dpi and to < 0.2% on 9 dpi. In all four but every attempt to get mosquitoes to refeed failed, de- canaries, only two or fewer P. homopolare gametocytes were spite providing an opportunity for the mosquitoes to ovi- visualized per 1000 erythrocytes (≤ 0.002%) on days 5, 7, and posit eggs developed after their primary (first infective) 9 post infection. It was possible that some trophozoites that we blood meal. Therefore, we were forced to use an artificial called P. cathemerium may have been P. homopolare tropho- capillary tube method (Aitken 1977;Corneletal. 1993)to zoites, because it is very difficult to differentiate trophozoites test for sporozoites secreted in saliva extrapolates at 15, 20, of the two species morphologically (Valkiūnas 2005). All ca- and 25 dpi. Initially, we filled each capillary with a solu- naries survived the experimental infection, and only after tion containing equal parts of heat-inactivated fetal bovine 2 months did they test positive for P. homopolare by PCR. serum (FBS; Sigma-Aldrich, St. Louis, MO) and 10% su- Microscopically, trophozoites of P. homopolare were still vis- crose. However, after constant parasite-negative results, ible in the blood, indicating that a chronic infection persisted. despite using mosquitoes with known parasite-positive sal- P. cathemerium was no longer detected by PCR or seen in ivary glands, we concluded that this method had to be blood smears after 2 months post-infection. Infection with a modified for Plasmodium studies. We then used a modified third parasite lineage became apparent xenodiagnotically only version of the Rosenberg et al. (1990) method, which did when the experimentally infected mosquitoes were tested provide PCR-positive saliva samples. In this method, the for parasite DNA. This third parasite had a cyt b sequence fascicle sheath of the mosquito was first removed and the identical to P. cathemerium lineage HOFI_CA_ELW_8P exposed proboscis was then inserted into the capillary (Pcat8; GenBank accession number KJ620781) that was tubes filled with a mixture of one part mineral oil and previously reported by Carlson et al. (2015)asa P. one part 10% sucrose. The mosquito was allowed to ex- cathemerium-like lineage isolated from mosquitoes and pectorate for 15 min before being removed for further birds in China Creek Park. Pcat6 and Pcat8 lineages have processing. a genetic distance of 0.85%. Parasitol Res (2018) 117:2385–2394 2389 Mosquito infections significantly among mosquito species based on the logistic regression with adjustment for the trend over time post- A total of 286 mosquitoes fully blood-fed on Plasmodium infection (Fig. 3). For Cx. stigmatosoma, the mean probability infected canaries B and D between 5 and 25 dpi. Of these of Pcat6 and Pcat8 salivary gland infection at 20 dpi was blood-fed mosquitoes, 115 were dissected at 15, 20, and 25 39.2% (95% CI 20.4–61.9%). The probability of infection dpi (Table 1), of which 44 were infected with Plasmodium was significantly lower for Cx. pipiens at 2.7% (P =0.0004; (37%). The other 169 blood-fed mosquitoes died before or 95% CI 0.7–10.5%) and highest for Cx. tarsalis at 79.0% (P = in between the time points and could not be dissected, but 0.0063; 95% CI 58.5–91.0%) at 20 dpi. were preserved in 70% ethanol. The majority (153/169) of Detection of sporozoites was attempted by collecting ex- the mosquitoes that died were Cx. tarsalis that had been col- pectorate samples by the capillary tube method. Only three lected from the field. Of the 169 dead mosquitoes, 66 (39%) saliva samples collected by the modified Rosenberg et al. thoraces were positive when tested by PCR. Table 1 shows the (1990) method described above tested positive by PCR. Two differences between the feeding patterns on canaries B and D. Cx. stigmatosoma and one Cx. tarsalis were positive for line- Cx. stigmatosoma did not readily take a blood meal from age Pcat8. canary B, despite having equal opportunity to feed on this Oocysts were counted in the midguts from each mosquito canary as other mosquito species. at each of the three post-infection time points. Total number of Plasmodium homopolare was not detected in any of oocysts ranged from 1 to 23 in Cx. stigmatosoma (n =16), the blood-fed mosquitoes. Fig. 2 shows the percentage of un- from 1 to 15 in Cx. tarsalis (n = 22), and from 1 to 28 (n = infected vs infected females for each species and the percent- 6) in Cx. quinquefasciatus. The single Cx. pipiens- age infection with the two P. cathemerium lineages. P. quinquefasciatus hybrid mosquito that tested positive had cathemerium lineage Pcat6 was detected in salivary glands three oocysts. Because it is impossible to morphologically of all species except Cx. pipiens, whereas lineage Pcat8 was distinguish among Plasmodium species during the oocyst detected in only Cx. stigmatosoma and Cx. tarsalis. The prob- and sporozoite stages (Valkiūnas 2005), we were not able to ability of sporozoite infection by P. cathemerium parasites determine whether these oocysts were from one or more (based on salivary gland infections by PCR) differed species. One potential solution for this in future studies Table 1 Table 1 reports mosquito infections tested on 15, 20, and 25 dpi For each of the five mosquito species tested, the total number of resulting from blood meals obtained from canary B and canary D, positive thoraxes (T) and salivary glands (S) are reported, followed by respectively. For each of the three time points, the infections are the total sample size (N). The symbol B-^ characterizes samples for which reported for each of the two Plasmodium cathemerium lineages Pcat6 a PCR was not carried out because there were not enough mosquito (SPTO_CA_ELW_6P, GenBank accession number KJ620779) and specimens to test at that time point Pcat8 (HOFI_CA_ELW_8P, GenBank accession number KJ620781). Canary B Mosquito species Plasmodium cathemerium Cx. pipiens Cx. quinquefasciatus Cx. quinquefasciatus-pipiens Cx. stigmatosoma Cx. tarsalis lineage hybrids DPI T (+) S (+) NT (+) S (+) NT (+) S (+) NT (+) S (+) NT (+) S (+) N 15 SPTO_CA_ELW_6P - - 0 - - 0 - - 0 - - 0 3 1 3 HOFI_CA_ELW_8P - - - - - - - - 0 0 20 SPTO_CA_ELW_6P 0 0 2 2 0 10 1 1 7 - - 0 1 1 1 HOFI_CA_ELW_8P 0 0 0 0 0 0 - - 0 0 25 SPTO_CA_ELW_6P 0 0 2 3 0 10 0 0 4 - - 0 1 1 2 HOFI_CA_ELW_8P 0 0 0 1 0 0 - - 1 1 Total 0 04 5 1 20 1 1 11 - - 0 6 46 Canary D Mosquito species Plasmodium cathemerium Cx. pipiens Cx. quinquefasciatus Cx. quinquefasciatus-pipiens Cx. stigmatosoma Cx. tarsalis lineage hybrids DPI T (+) S (+) NT (+) S (+) NT (+) S (+) NT (+) S (+) NT (+) S (+) N 15 SPTO_CA_ELW_6P 0 0 1 0 0 10 0 0 4 2 1 12 7 6 10 HOFI_CA_ELW_8P 0 0 0 0 0 0 8 4 0 0 20 SPTO_CA_ELW_6P - - 0 0 0 7 0 0 2 0 3 7 6 6 8 HOFI_CA_ELW_8P - - 0 0 0 0 5 0 1 1 25 SPTO_CA_ELW_6P - - 0 0 0 6 0 0 2 0 0 3 1 1 2 HOFI_CA_ELW_8P - - 0 0 0 0 1 0 1 1 Total 0 0 1 0 0 23 0 0 8 16 8 22 16 15 20 2390 Parasitol Res (2018) 117:2385–2394 Fig. 2 Prevalence (reported as total % of all mosquitoes tested at 15, 20, mosquito species tested. No mosquitoes were infected with P. and 25 dpi) of Plasmodium cathemerium lineages Pcat6 (SPTO_CA_ homopolare. No infections were observed in Cx. pipiens. Cx. tarsalis ELW_6P, GenBank accession number KJ620779) and Pcat8 (HOFI_ presented the highest prevalence of all five species. P. cathemerium CA_ELW_8P, GenBank accession number KJ620781) in the five lineage Pcat8 was only detected in Cx. stigmatosoma and Cx. tarsalis could be to use laser capture microdissections of midgut low levels of < 0.002%. Non-exclusive hypotheses may be epithelia of mosquitoes, similar to the methods described suggested as following: (1) this level of gametocytemia was by Lutz et al. (2016). too low for effective syngamy within the mosquito midguts precluding the infection of any species of mosquito; (2) co- infection with P. cathemerium prevented P. homopolare from Discussion developing an infection in the mosquito; (3) the mosquito species in this study are not the natural vector of P. Experimental infection homopolare; and/or (4) the PCR assay was not sensitive A question often asked in disease ecology is as follows: what determines pathogen infection dynamics in time and space to explain trends in maintenance and spread? It is generally agreed that the main determinants of the structuring of parasite and host associations and heterogeneity of infection in a host population are host exposure and innate and adaptive immune responses to the pathogens (Poulin 2011). Host exposure is influenced by the capacity of the local vectors to transmit the pathogens, otherwise known as vectorial capacity (Garrett- Jones and Shidrawi 1969). In studies conducted at China Creek Park (Carlson et al. 2015; Walther et al. 2016), incon- gruence between host and vector Plasmodium infection prev- alences led to proposing that specific parasite-vector interac- tions (incompatibilities and compatibilities) were likely occur- ring, which could be tested by vector experimental infection assays. Experimental vector infection studies undertaken in Fig. 3 The probability of sporozoite infection by Plasmodium parasites (based on prevalence of salivary gland infections using logistic regression this study revealed that mixed Plasmodium infections, which with adjustment for the trend over time post-infection for Culex pipiens are common in hosts (Manwell and Herman 1935;Dimitrov et complex, Cx. stigmatosoma,and Cx. tarsalis. Bars represent the 95% al. 2015; Palinauskas et al. 2011), raise new interpretative confidence intervals surrounding the mean probability of infection. The challenges and parasite species interactions in vectors, which mean probability of infection at 20 dpi was 39.2% (95% CI 20.4–61.9%) for Cx. stigmatosoma. The probability of infection was significantly low- can determine relative parasite abundance in time and space. er for Culex pipiens complex at 2.7% (P = 0.0004; 95% CI 0.7–10.5%) One of the obvious results in this study was that none of the and significantly higher for Cx. tarsalis at 79.0% (P = 0.0063; 95% CI mosquitoes presented with P. homopolare became infected, 58.5–91.0%) compared to that for Cx. stigmatosoma;**P <0.01, ***P < 0.001 despite imbibing gametocytes of this species, admittedly at Parasitol Res (2018) 117:2385–2394 2391 enough to detect P. homopolare when it was simultaneously considered generalists with the ability to infect multiple distant- present with the two P. cathemerium lineages. ly related host taxa and can switch between resident and mi- grant bird species (Waldenström et al. 2002). Little is known 1: P. homopolare was described in 2014 (Walther et al. about the transmission cycle of P. homopolare, especially in the 2014), and little is known about the life cycle and pathol- sense of its course of infection of the acute phase vs the chronic ogy of this species. Considering the extremely high prev- phase. Thus, we had no a priori knowledge on how this parasite alence but low parasitemia in the birds captured at China would infect our laboratory canaries; in other words, we do not Creek (< 0.25% parasitemia, Walther et al. 2016)and in know if canaries can be naturally infected with this parasite. the canaries in this study (< 0.002%), it is possible that P. However, it has been reported in the human malaria field that P. homopolare progresses to a long-lasting chronic infection vivax is suppressed during an acute infection in the presence of in hosts and there is only a short window of time during P. falciparum, but re-emerges as a chronic infection once P. which the gametocytemia is high enough to infect mos- falciparum subsided (Boyd and Kitchen 1937; Maitland et al. quitoes. In this study, we were unable to determine the 1996; Bruce et al. 2000). This could be explained by immune- minimum gametocytemia level required for P. mediated apparent competition in a host where there are mod- homopolare to infect mosquitoes, and we may have ifications to host susceptibility when a host’s immune response missed the infectious window. to one parasite affects its ability to control a second species. 2: Competition with congeneric parasites limits P. homopolare infections in mosquitoes. There are opportu- 3: The vector responsible for maintenance of P. homopolare nities for Plasmodium species to interact in mosquitoes, in nature was not among the species used in our study. At especially when vector species overlap (Paul et al. 2002) China Creek Park, Carlson et al. (2015) reported that only and mixed infections in infective birds are quite common 3 out of the 76 Plasmodium-positive field-collected mos- (Valkiūnas et al. 2003; Biedrzycka et al. 2015). In in vitro quito thoraxes were infected with P. homopolare:one Cx. studies, Valkiūnas et al. (2013, 2014) noted several repro- tarsalis (which was the only individual with positive sal- ductive outcomes in blood containing gametes from sever- ivary glands), one Cx. restuans, and one Culiseta al avian Haemoproteus parasite species. Outcomes includ- particeps. However, at the same site, Walther et al. ed complete blockage of development of ookinetes of (2014) reported that 68% of infected birds were infected some species, to increase in reproductive success (increase with P. homopolare by PCR, but not all blood smears in ookinete production) of other species and, on occasion, were checked for gametocytes. production of hybrid ookinetes. Based on our infective studies, it is possible that P. cathemerium blocked syngamy Combes (1991) described the two ecological drivers of het- and development of P. homopolare ookinetes in the species erogeneous distribution of parasites in a host population as the of mosquitoes that were infected. Much knowledge is lack- α and β filters. The encounter filter α refers to the ecological ing concerning in vivo interactions of Plasmodium gam- and/or behavioral obstacles that result in the exclusion of a etes and ookinete development in mixed infections in mos- parasite in a host species. The compatibility filter β refers to quitoes, studies of which will be enhanced, when parasite the successful metabolic and/or immunological response in an species-specific nuclear markers are available which can individual host to an invading pathogen that results in the ex- detect hybrids (Valkiūnas et al. 2014). clusion of species that do not permit coexistence with the in- vading pathogen. Very few studies have tested the α and β Moreover, when discussing parasite-parasite competition, it filters directly in avian malaria vectors. According to several is also important to consider host specificity in the context of prior studies (Gager et al. 2008;Njabo etal. 2011; Medeiros et parasites and the relationship between host breadth and host- al. 2013;Valkiūnas et al. 2015), vectors do not play a role in use proficiency. P. homopolare was recently described by driving avian Plasmodium parasite ranges and that ranges are Walther et al. (2014) as a new species and P. cathemerium is determined by host compatibilities. This may not be universally a generalist parasite, having been detected in 9 families and 26 true, and the range of P. homopolare maybedriven by the species, with a worldwide distribution (Valkiūnas 2005). P. presence of compatible vectors such as Cx. restuans in the homopolare infected 84 birds of 399 birds collected at China northern hemisphere. Perhaps, there is specific vector compat- Creek Park, representing 9 host species from 5 families. A total ibility of P. homopolare for Cx. restuans. Cx. restuans is wide- of 31% of birds collected at China Creek Park were infected spread in the USA but also has a patchy distribution (Darsie and with Plasmodium, and 68% were infected with P. homopolare, Ward 2005) with preferences for marshy areas. Several other which is the same as saying that 1 in 5 birds at China Creek studies provide evidence for a β filter by demonstrating Park were infected with P. homopolare (Walther et al. 2014).P. genotype-by-genotype interactions between pathogens and cathemerium was found in 6 bird species and 3 families at their vectors, which then may mean that the structuring China Creek Park. This means that both parasites are of pathogens within populations may be, in part, a result 2392 Parasitol Res (2018) 117:2385–2394 of adaptation of pathogens to local vector genotypes species, should use a combination of transmission assays and (Ferguson and Read 2002; Schmid-Hempel and Ebert multiple Plasmodium species-specific primers on various 2003; Lambrechts et al. 2005;Joy etal. 2008; mosquito parts and extracts especially to identify potentials Lambrechts et al. 2009). For example, Lambrechts et al. of co-infections/interactions and transmissibility. (2009) provide experimental evidence for the potential role of vector-driven genetic structuring of dengue viruses. Consequences of mixed infections and vector Clearly, for avian malaria, more studies are needed to discern compatibility the role of vectors as potential filters. Boëte and Paul (2006) stated that current species dynamics of 4: The apparent absence of P. homopolare in laboratory- parasites could become disturbed by control measures and infected mosquitoes might have resulted from deficien- may lead to epidemiological changes, but the predictability cies in current diagnostic tools (Bernotienė et al. 2016; of the changes would be dependent on the level of equilibrium Clark et al. 2016). Only after several host passages and within the system. Because there are multiple levels in which vector infectivity studies did we discover that the original parasites can interact with one another through competition, donor song sparrow was infected with three Plasmodium such as resource, interference, and immune-mediated compe- species and lineages. The determination of the infection tition, it is not clear at which point these parasites reach a state status in the host and the vector can be done by micros- of equilibrium, if ever at all (Snounou and White 2004). copy, ELISA, and PCR-based methodologies, although As mentioned above, the general consensus is that hosts each method presents challenges. There is limited world- rather than the vectors drive avian Plasmodium parasite geo- wide expertise available to morphologically identify the graphical ranges (Medeiros et al. 2013). However, in our field erythrocytic stages of parasites, and this is particularly system, it is not clear how P. homopolare was able to be so difficult when infections are at the early trophozoite abundant in the resident avian population. The results from stages and at low parasitemias. In mosquito slide prepa- our vector competence assay indicated that there needs to be rations, it is impossible to morphologically distinguish more research conducted on how competition among parasites between parasite species. ELISA and PCR-based method- may be driving how mosquitoes transmit the parasites. There ologies can detect conspecific infections provided is support for this notion in the context of mixed infections. species-specific circumsporozoite (CS) proteins and Paul et al. (2002) proposed that interspecific competition dur- primers are available (Coleman et al. 2002;Marchand et ing transmission in the vector may have contributed to a re- al. 2011). Species-specific CS protein and primers are not striction of P. gallinaceum around the world. Presence of P. available for most avian malaria parasites, and we propose juxtanucleare may reduce the R of P. gallinaceum and could that co-infections of avian Plasmodium are therefore un- reduce the invasion or establishment of P. gallinaceum. derrepresented in host and vector prevalence studies. Further supporting evidence can be found in examples of Additionally, the PCR primer set used in this study, which competition for red blood cells affecting parasitemias was not species-specific, has been shown to preferentially (McQueen and Mckenzie 2006) and gametocyte production amplify one parasite species or lineage over another in the (Bousema et al. 2008) which will alter the potential for estab- presence of co-infections (Zehtindjiev et al. 2012). These lishing infections in mosquitoes. Because of mixed infections vector infectivity studies confirmed that mosquitoes can complicating outcomes of the vector competence trials in this support co-infections as the thorax and the salivary glands study, we were unable to make tacit conclusions about the from individual mosquitoes were infected with different susceptibilities of mosquitoes tested for P. homopolare. lineages of P. cathemerium simultaneously. If vector compatibility differences do occur, heterogeneity in parasite ranges and temporal occurrence can be expected. Unfortunately, we were unsuccessful in coaxing the infect- Different prevalences of P. vivax phenotypes exist between ed mosquitoes to take a second blood meal on naïve canaries Gulf of Mexico and Pacific coastlines because of differences to test whether they were capable of transmitting multiple in susceptibilities and geographic distributions between Plasmodium to a recipient canary. The artificial capillary Anopheles albimanus (favoring phenotype VK210) and An. method that was used to detect the expectoration of sporozo- pseudopunctipennis (favoring phenotype VK247) (Rodriguez ites showed some promise. However, this method likely needs et al. 2000). Temporal fluctuations of these two P. vivax phe- considerable refinement and testing before it can be used as a notypes were also noted in Thailand, and one of the explana- reliable surrogate method for testing in vivo transmission of tions offered was related to seasonal abundance of susceptible avian sporozoites. In addition, suitable PCR methods must be vectors (Suwanabun et al. 1994). P. homopolare is likely quite available to determine which Plasmodium species sporozoites widespread in the USA, based on 100% sequence matches in have been expectorated. Future avian vector competence stud- GenBank from various bird species (Walther et al. 2014), but ies, especially when performed on non-cultured Plasmodium on more local scales has a patchy distribution. In California, Parasitol Res (2018) 117:2385–2394 2393 characterization of blood parasites in a sedge warbler population no P. homopolare parasites were seen in blood smears or de- from southern Poland. J Ornithol 156:201–208 tected by PCR in 200 birds in 2014 and 2015 at the Stone Boëte C, Paul REL (2006) Can mosquitoes help to unravel the commu- Lakes NWR, Elk Grove, CA (Carlson et al. unpublished data). nity structure of Plasmodium species? Trends Parasitol 22:21–25 Both China Creek and Stone Lakes share similar riverine hab- Bousema JT, Drakeley CJ, Mens PF, Arens T, Houben R, Omar SA, Gouagna LC, Schallig H, Sauerwein RW (2008) Increased itat, bird species, and mosquito species except for Cx. Plasmodium falciparum gametocyte production in mixed infections restuans, which were not found in the latter site. with P. malariae. Am J Trop Med Hyg 78:442–448 Vector competence for avian malaria parasites in the Cx. Boyd MF, Kitchen SF (1937) Simultaneous inoculation with Plasmodium pipiens complex in California should be further studied. It is Vivax and Plasmodium falciparum. Am J Trop Med Hyg 1:855–861 Bruce MC, Donnelly CA, Alpers MP, Galinski MR, Barnwell JW, curious that along with this study, reports by Carlson et al. Walliker D, Day KP (2000) Cross-species interactions between ma- (2015) and by Carlson et al. (2016)identify Cx. pipiens com- laria parasites in humans. Science 287:845–848 plex as a low importance vector. Because Kothera et al. (2013) Carlson JS, Walther E, Trout Fryxell R, Staley S, Tell LA, Sehgal RNM, propose that Cx. pipiens are mostly hybrids of Cx. pipiens and Barker CM, Cornel AJ (2015) Identifying avian malaria vectors: sampling methods influence outcomes. Parasit Vectors 8:365 Cx. quinquefasciatus in California, it is plausible that this Carlson JS, Giannitti F, Valkiūnas G, Tell LA, Snipes J, Wright S, Cornel genetic makeup allows them to be less permissible to avian AJ (2016) A method to preserve low parasitaemia Plasmodium-in- malaria parasites in comparison to most reports on these spe- fected avian blood for host and vector infectivity assays. Malar J 15: cies elsewhere in the world (Valkiūnas 2005). 154 Clark NJ, Wells K, Dimitrov D, Clegg SM (2016) Co-infections and environmental conditions drive the distributions of blood parasites Acknowledgements We are thankful to the Lake County Vector Control in wild birds. J Anim Ecol 85:1461–1470 District, the Consolidate Abatement District of Fresno County, the Sutter- Coleman RE, Sithiprasasna R, Kankaew P, Kiattibut C, Ratanawong S, Yuba Mosquito and Vector Control District, and the Greater Los Angeles Khuntirat B, Sattabongkot J, (2002) Naturally Occurring Mixed County Vector Control District for providing mosquitoes. We would also Infection of VK210 and VK247 in Mosquitoes (Diptera: like to extend our gratitude to the Center of Vectorborne Diseases who Culicidae) in Western Thailand. J Med Entomol 39 (3):556–559. granted us access to the BSL 3 facilities where both canaries and mos- Combes C (1991) Evolution of parasite life cycles. In Parasite–host as- quitoes could be simultaneously kept for the duration of the experiment. sociations: coexistence or conflict, Toft C, Aeschlimann A, Bolis L, Plasmodium bird housing and infectivity protocols complied with Editors. Oxford University Press: Oxford. p. 62–82 IACUC permit 17601 issued at the University of California at Davis Cornel AJ, Jupp PG, Blackburn NK (1993) Environmental temperature (UC Davis). on the vector competence of Culex univittatus (Diptera: Culicidae) for West Nile virus. J Med Entomol 30:449–456 Funding information This work was supported by the UC Davis Henry Cummings RF (1992) Design and use of a modified Reiter gravid mos- A. Jastro Research Award, by the UC Davis Bill Hazeltine Student quito trap for mosquito-borne encephalitis surveillance in Los Research Award, and by the Mosquito Research Foundation. Angeles County, California. Proc Papers Mosq Vector Control Assoc Calif 60:170–176 Compliance with ethical standards Darsie RF, Ward RA (2005) Identification and geographical distribution of the mosquitoes of North America, North of Mexico. University Competing interests The authors declare that they have no competing Press of Florida, Gainesville interests. 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