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The consequences of mixed-species malaria parasite co-infections in mice and mosquitoes for disease severity, parasite fitness, and transmission success

The distributions of human malaria parasite species overlap in most malarious regions of the world, and co-infections involving two or more malaria parasite species are common. Little is known about the consequences of interactions between species during co-infection for disease severity and parasite transmission success. Anti-malarial interventions can have disproportionate effects on malaria parasite species, and may locally differentially reduce the number of species in circulation. Thus, it is important to have a clearer understanding of how the interactions between species affect disease and transmission dynamics. Controlled competition experiments using human malaria parasites are impossible, and thus we assessed the consequences of mixed-species infections on parasite fitness, disease severity, and transmission success using the rodent malaria parasite species Plasmodium chabaudi, P. yoelii yoelii, and P. vinckei lentum. We compared the fitness of individual species within single species and co-infections in mice. We also assessed the disease severity of single versus mixed infections in mice by measuring mortality rates, anaemia, and weight loss. Finally, we compared the transmission success of parasites in single or mixed species infections by quantifying oocyst development in Anopheles stephensi mosquitoes. We found that co-infections of P. yoelii with either P. vinckei or P. chabaudi led to a dramatic increase in infection virulence, with 100% mortality observed in mixed species infections, compared to no mortality for P. yoelii and P. vinckei single infections, and 40% mortality for P. chabaudi single infections. The increased mortality in the mixed infections was associated with an inability to clear parasitaemia, with the non-P. yoelii parasite species persisting at higher parasite densities than in single infections. P. yoelii growth was suppressed in all mixed infections compared to single infections. Transmissibility of P. vinckei and P. chabaudi to mosquitoes was also reduced in the presence of P. yoelii in co-infections compared to single infections. The increased virulence of co-infections containing P. yoelii (reticulocyte restricted) and P. chabaudi or P. vinckei (predominantly normocyte restricted) may be due to parasite cell tropism and/or immune modulation of the host. We explain the reduction in transmission success of species in co-infections in terms of inter-species gamete incompatibility.


Introduction
Eight malaria parasite species are infectious to humans; namely, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale wallikeri, Plasmodium ovale curtisii, Plasmodium knowlesi, Plasmodium cynomolgi, and Plasmodium simium. The latter three species are parasites of non-human primates, but also cause zoonotic malaria in humans [1][2][3]. In large parts of the tropical world the ranges of at least some of these species overlap, they are often vectored by the same mosquitoes [4], and mixed-species infections are common [5][6][7].
The consequences of mixed-species infections on malaria disease and parasite fitness are incompletely understood. There is conflicting evidence from laboratory and field studies regarding the capacity of mixed-species infections to exacerbate [27] or ameliorate [14] disease. Furthermore, the mechanisms underlying the interactions between parasite species in mixed infections are complicated and multi-factorial, possibly involving both within-host competition [28] and cross-immunity [29].
Mixed species and mixed strain Plasmodium infections have been studied in primate [30] and rodent malaria parasite models [27,28,31,32], as these enable the study of all parasite lifecycle stages including those that occur in mosquitoes. The rodent model is enhanced by the availability of multiple rodent malaria parasite species; namely, Plasmodium yoelii, Plasmodium berghei, Plasmodium vinckei, and Plasmodium chabaudi. For P. yoelii there are additionally several parasite strains that differ in virulence following inoculation of mice [33].
Here we describe the results of a series of experiments utilising multiple strains of the rodent malaria parasite species P. yoelii, P. chabaudi, and P. vinckei, in which mixed infections of various combinations of species and strains were established and studied in both mice and mosquitoes. The consequences of mixed species infections for disease severity in both hosts, parasite fitness, and transmission capacity were analysed.

Ethics statement
Animal experimentation was performed in strict accordance with the Japanese Humane Treatment and Management of Animals Law (law No. 105, 1973;modified 2006), and according to the Regulations on Animal Experimentation at Nagasaki University, Japan. All procedures were approved by the Institutional Animal Research Committee of Nagasaki University.
Six-week-old female CBA mice, Mus musculus, were purchased from SLC Inc. (Shizuoka, Japan) and were used for all experiments. Mice were housed in a 12-hour/12-hour light/dark cycle at °C and fed with 0.05% para-aminobenzoic acid (PABA)-supplemented water to assist the growth of parasites. Anopheles stephensi mosquitoes were housed in a temperature-and humidity-controlled insectary at 23°C and 75% humidity. Mosquitoes used in the transmission experiments were maintained on 10% glucose solution supplemented with 0.05% PABA.

DNA extraction and real time quantitative PCR (qPCR)
To determine the proportion of each species in mixed infections, quantitative real time PCR (qPCR) was used to measure copy numbers of the merozoite surface protein 1 gene (msp1).
DNA was extracted from infected mouse tail blood and infected mosquito midguts using an EZI DNA Investigator Kit (GIAGQN) according to the manufacturer's instructions. Quantitative PCR was performed on an ABI 7500 real-time PCR machine using a Power SYBR Green kit (Applied Biosystems, UK). Primers were designed based on a species-specific region of msp1, as follows:

Mosquito transmission experiments: estimation of mosquito fitness and parasite species transmission capacity
Groups of mice were infected with single and mixed species infections of PyCU, PvDS, and PcAJ parasites (total 6 groups, each of 5 mice). On days 3 and 5 post-inoculation, individual groups of mosquitoes (n = 40 mosquitoes per group; 5-7 days post emergence from pupae) were fed on individual mice. Immediately following the feed, 20 mosquitoes from each group were pooled by mouse group into 12 cages, and egg bowls added two days later to allow the collection of eggs. These groups were monitored for longevity by counting dead mosquitoes daily up to day 60, and the numbers of larvae produced per mosquito were counted at day 5 post-hatching. Seven days later, 20 mosquitoes were removed from each group (57 groups total, as the number of mice fed from group PvDS and PcAJ+PvDS was reduced to three and four, respectively for the day 5 feed) and the midgut oocyst burden recorded following dissection. Dissected midguts were stored at -80°C prior to DNA extraction for species proportion analysis by qPCR.

Statistical analyses
All graphs were generated using GraphPad Prism 6 (GraphPad software Inc, USA P-values of below 0.05 were considered significant.

Results
Mixed species parasite infections involving a reticulocyte specialist and a normocyte specialist are more virulent and cause greater host mortality than single species infections in mice Infection parameters for single and mixed infections involving PyCU (reticulocyte restricted) and either PvDS, PcAJ, or PcASED (normocyte preference) are summarized in Table 1. to the inability of mice to control parasitaemia.

Mixed species parasite infections involving two normocyte specialists result in protracted
parasitaemia, but not increased virulence P. vinckei DS and P. chabaudi AJ are both normocyte invading parasites. PcAJ is of moderate virulence, causing rapid and severe anaemia and weight loss during the first 10 days of infection when parasitaemia rises, and results in 40% host mortality ( Table 2 and Fig. 2A).
PvDS is a much less virulent parasite, causing less severe weight loss and milder anaemia, and is never lethal ( Table 2 Fig. 2A). The combination of these two parasites in a mixed infection results in 50% host mortality, and a pathology consistent with that of PcAJ, the more virulent of the two species (Table 2

Pre-exposure of mice to P. yoelii does not enhance the virulence of P. vinckei infection
As the presence of P. yoelii in mixed infections with either P. chabaudi or P. vinckei results in an inability to clear the latter two species, resulting in host death, we wondered whether an immune response specific to P. yoelii could adversely affect the establishment of an effective immune response against P. chabaudi or P. vinckei. To test this, we pre-immunised mice with PyCU parasites by exposure to the parasite for eight days followed by clearance with the antischizontal drug mefloquine (MF). Two weeks later, when all MF had cleared from the host, mice were challenged with PvDS. In contrast to the patterns observed in PyCu+PvDS coinfections, there was no evidence of increased virulence of PvDS infections in mice preexposed to PyCU (Fig. 4A), with no significant enhancement of parasite density (Fig. 4B), anaemia (Fig. 4C), or weight loss (Fig. 4D) occurring at any stage during the infection.

The consequences of mixed species infections in the mosquito vector
We additionally sought to describe the impact of mixed species infections on transmission to mosquitoes. Specifically, we fed Anopheles stephensi mosquitoes on mice with single or mixed species infections and measured: i) the proportion subsequently infected, and ii) the severity of this infection (number of oocysts); iii) the longevity of infected mosquitoes; and iv) the number of larvae they produced following a blood meal. Finally, we compared the transmission success, defined as the average number of oocysts produced per blood fed mosquito, of each parasite species in mixed or single infections.

Mixed species infections do not result in significantly different infection parameters in mosquitoes
To determine whether mixed species infections result in altered mosquito infectivity rates and infection loads compared to single species infections, mosquitoes were fed on anaesthetized mice infected with single, or mixed infections of PcCU, PvDS, and PcAJ. As these species differ in the timing of their gametocyte production, with PyCU at its most infectious to mosquitoes on day 3 PI and PcAJ and PvDS more infectious on day 5 pi, we conducted mosquito feeds on both these days. PyCu was the most infective single species on day 3 and day 5, followed by PvDS and finally PcAJ (Fig. 6A-C). Mixed species infections did not result in higher proportions of mosquitoes being infected than the most infective constituent species in a single infection (Fig. 6A-C). Similarly, in co-infections containing the highly infectious PyCU species, the oocyst burdens of mixed species infections were not significantly different from that of PyCU in mosquitoes fed on mixed infections on either day 3 or day 5 PI (Fig. 6D+E).
Mixed species infections of PcAJ and PvDS resulted in significantly lower oocyst burdens than the most infectious constituent single species (PvDS), but only in mosquitoes fed on day 5 of the infection (Fig. 6F).

Co-infections of malaria parasite species do not adversely affect mosquito longevity or capacity to produce larvae
To ascertain whether mixed species infections of mosquitoes were more virulent than single species infections, we measured longevity and larvae production in mosquitoes fed on single or mixed infections. In accordance with the observation that mixed species infections did not result in higher burdens of infection, mosquitoes infected with two parasite species in a co- 13 infection did not display reduced longevity (Fig. 7A-C), median survival time (Fig. 7D), or reduced fitness (measured as the number of larvae produced per blood-fed mosquito) (Fig.   7E).

Mixed species infections can affect the transmission capacity of the constituent species
To determine whether mixed infections can affect the transmission capacity of constituent species, the relative proportion of each species in mixed infections in mosquitoes was measured using qPCR on DNA extracted from mosquitoes with known oocyst numbers and compared to the numbers produced in single infections.
The numbers of oocysts produced by the highly infectious PyCU did not differ significantly between mosquitoes fed on single and mixed species infections (Fig. 8A). PvDS produced fewer oocysts in mixed infections with PcAJ and PyCU than in single infections, although the effect was only statistically significant on day 5 in a mixed infection with PyCU (Fig. 8B). PcAJ also suffered a reduced transmission capacity in mixed infections, with significant reductions in oocyst numbers measured in mosquitoes fed on mixed infections with either PvDS or PyCU on day 5, compared to those fed on single infections (Fig. 8C). The transmission of PcAJ to mosquitoes was completely blocked in mixed infections containing PyCU (Fig. 8C).

Discussion
The major malaria parasite species that naturally infect man have overlapping ranges in many tropical regions, the greatest exception being caused the apparent absence of P. vivax from large parts of west and central and central Africa, where P. ovale, P. malariae and P.
falciparum are at their most abundant. It is often difficult to interpret data regarding the consequences of the mixed-species malaria infections that occur naturally in man, due to the multitude of confounding factors that exist in nature.
Our results indicate that the interactions between malaria parasites co-infecting the same host can have dramatic consequences for the severity of the disease they cause. We found that when two parasite species, P. yoelii and P. vinckei, which on their own cause mild and transient disease concurrently infect the same host, the disease outcome is radically altered resulting in 100% host mortality within 15 days. This same outcome of 100% host mortality was observed in co-infections consisting of P. yoelii and the more virulent (but rarely lethal)

P. chabaudi.
There are precedents for this result; Bafort (1971) suggested that mixed species might increase the virulence of infections [36]. Richie (1985) reported that patent P. chabaudi infections increased their parasitaemia and duration when mixed with P. yoelii [9], and McGhee (1964) also described a higher peak for one of the species in mixed infections [37].
More recently, Ramiro et al (2016) described increased virulence in mixed infections of P.
chabaudi and P. yoelii, which they attributed to an increase in reticulocytaemia leading to enhancement of P. yoelii (which is reticulocyte restricted) in the mixed infections. Our results, however, are in agreement with those of Richie (1988), and suggest that it is the normocyterestricted parasite that is enhanced in mixed infections with P. yoelii, a result we observed both in the case of two strains of P. chabaudi and one of P. vinckei. We contend, therefore, that increasing reticulocytaemia does not explain the increase in virulence of mixed strain infections. This is supported by the fact that we also observed increased virulence (in terms of persistence of infection and decreased red blood cell density) in mixed-species infections composed of P. chabaudi and P. vinckei, both of which predominantly infect normocytes.
We found that the time at which the constituent species of a mixed-species infection were introduced to the host had a significant impact on disease outcome. Most studies on mixed malaria parasite species infections in mice introduce the constituent species contemporaneously [27, 28]. For example, the parasite species combination P. yoelii and P.
vinckei causes 100% mortality when introduced to mice contemporaneously. We found that when P. yoelii was inoculated seven days prior to the inoculation of P. vinckei, virulence was much reduced, although the mixed infection still caused significantly more pathology and mortality than the constituent species in single infections. Similarly, when inoculated into mice contemporaneously with P. vinckei, P. yoelii suffered a reduction in cumulative parasite density throughout the infection (a proxy measurement of parasite fitness), whereas P.
vinckei was unaffected. When P. yoelii was introduced to mice a week before P. vinckei, the opposite trend was observed, with little reduction in the cumulative parasite density of P.
yoelii, but a significant reduction in that of P. vinckei. We show, therefore, that it is not just the phenotypes of the constituent species of mixed infections that affect pathology and parasite fitness in combination, but also the time at which each species infects the host. to the expectations of the original antigenic sin hypothesis, we observed no effect on the severity of the P. vinckei infection in P. yoelii-exposed compared to non-exposed mice.

It is possible that interactions between malaria parasite species in mixed infections
Infection with malaria parasites is known to detrimentally affect the fitness of the infected mosquito [44]. Mixed species malaria parasite infections are common in nature [4,45], and as mixed species parasite infections caused dramatically different disease outcomes in mice, we investigated whether mosquito fitness was also affected. We measured longevity and progeny production in groups of mosquitoes fed on single and mixed species infections. In contrast to the significant alterations in pathogenicity observed in mice, there appeared to be no fitness differences between mosquitoes carrying single or mixed species infections.
Linked to this, we did not observe significantly increased oocyst numbers in mosquitoes infected with mixed species, when compared to the highest-oocyst producing single constituent species, suggesting there was no significant alteration in transmission-stage investment by the species in mixed stage infections [31].
We found that the transmissibility of P. vinckei and P. chabaudi to mosquitoes was reduced in the presence of P. yoelii in co-infections compared to single infections. This was reflected in the lower number of oocysts of these two species in mosquitoes that had fed on mixed species infections also containing P. yoelii compared to those that had fed on single infections.
Most significantly, transmission of P. chabaudi to mosquitoes was blocked completely by the presence of P. yoelii on day 5 of the infection. There were no reductions, however, in the numbers of P. yoelii oocysts. Of the three species, P. yoelii produces significantly higher oocyst burdens in mosquitoes than either P. chabaudi or P. vinckei, a phenomenon linked to the former species having much higher gametocyte production during the early stages of infection. One possible mechanism that may account for this observation involves gamete incompatibility; we propose that given the fact that in a mixed species infection containing P.
yoelii, there will be significantly more P. yoelii microgametes than of the other species. If P.
yoelii microgametes can recognise and attempt to fertilise the macrogametes of the second species, then a large proportion of these macrogametes will be rendered non-productive (assuming hybrids are non-viable) [46]. Consistent with this theory is the fact that both P. chabaudi and P. vinckei also produce fewer oocysts when in mixed infection with each other, but much less so than when mixed with P. yoelii, reflecting, perhaps, the more even numbers of gametocytes produced by these two species.
As the world moves towards reducing the malaria burden, the importance of mixed-species malaria infections will rise. Diagnostic techniques with improved sensitivities are revealing a greater prevalence of P. ovale and P. malariae in P. falciparum endemic areas than previously thought [7]. Intervention strategies such as anti-vector programs, and the development and employment of new drugs and vaccine will often be more effective against one species of parasite than they will against others [47,48]. In regions where P. falciparum prevalence is decreasing, the prevalence of non-falciparum malaria parasite species often becomes more apparent, highlighting the importance of mixed-species infections. There is a need, therefore, to better understand how the interactions between malaria parasites species infecting the same host can impact disease progression and parasite fitness. Detailed statistical values relating to significance are given in Supporting Table 1. Experiments were repeated twice, data is from one representative experiment. . Mice were inoculated via intravenous injection of 1x10 6 infected red blood cells of either single or mixed species of the above parasites and followed for 30 days to determine mortality, live-body weight, erythrocyte density, and parasitaemia. Data points indicate the mean value for mice of each experimental group and error bars indicate the standard error of the mean (SEM). An asterisk represents statistically significant difference of mixed infections compared with both single infections (Multiple t-tests, with the assumption that all rows are sampled from populations with the same scatter and corrected for multiple comparisons using the Holm-Sidak method). Mice infected with mixed-infections developed statistically significantly higher parasitaemia on days 5 and 9 PI compared with both single infections (panel B  Similarly, the cumulative parasite density of PcAJ in single infections was significantly higher than in mixed infections with PyCU or PvDS (panel I). Detailed statistical values relating to significance are given in Supporting Table 1. Experiments were repeated twice, data is from one representative experiment. Asterisks indicate statistically significant differences between mice with mixed-species infections compared with both single infections (Multiple t-tests, with the assumption that all rows are sampled from populations with the same scatter and corrected for multiple comparisons using the Holm-Sidak method). Mice infected with mixed-infections lost significantly more weight during the latter stages of the infection than mice infected with single species infections (days 21-23 PI, panel D). The relative proportion of each species in mixed infections (panel E) was measured by qPCR quantification of the msp1 gene. The average copy number per iRBC was generated with reference to copy numbers and parasite densities of each single species infections on day 6 PI. The parasite densities of PyCU and PvDS in single or mixed infections are shown in panels F and G. A Mann Whitney test shows that the cumulative parasite density of PvDS in single infections is significantly higher than that of PvDS in a mixed infection with PyCU (panel I), whereas that of PyCU is unaffected when in a mixed infection with PvDS (panel H). Detailed statistical values relating to significance are given in Supporting Table 1.  Table 1. Boxplots indicate median survival and first and third quartiles, and whiskers are the same quartiles ± (1.5×interquartile range) (panel D). The numbers of larvae from infected mosquitoes with single or mixed infections were recorded and no significant difference was observed. Detailed statistical values relating to significance are given in Supporting Table 1. and Plasmodium chabaudi AJ (PcAJ) in single or mixed infections. The mean percentage of mosquitoes infected with oocysts were calculated following feeding on PyCU, PvDS, and PcAJ either in single or mixed infections in mice on days 3 and 5 PI (panels A-C). Only gravid mosquitoes were considered blood-fed and included in the analysis. Statistical analysis was performed using Mann Whitney tests. Detailed statistical values relating to significance are given in Supporting Table 1.  Table 1.