Experimental infections and co-infections with Leishmania braziliensis and Leishmania infantum in two sand fly species, Lutzomyia migonei and Lutzomyia longipalpis

Leishmaniases are neglected tropical diseases and Leishmania (Leishmania) infantum and Leishmania (Viannia) braziliensis are the most important causative agents of leishmaniases in the New World. These two parasite species may co-circulate in a given endemic area but their interactions in the vector have not been studied yet. We conducted experimental infections using both single infections and co-infections to compare the development of L. (L.) infantum (OGVL/mCherry) and L. (V.) braziliensis (XB29/GFP) in Lutzomyia longipalpis and Lutzomyia migonei. Parasite labelling by different fluorescein proteins enabled studying interspecific competition and localization of different parasite species during co-infections. Both Leishmania species completed their life cycle, producing infective forms in both sand fly species studied. The same happens in the co infections, demonstrating that the two parasites conclude their development and do not compete with each other. However, infections produced by L. (L.) infantum reached higher rates and grew more vigorously, as compared to L. (V.) braziliensis. In late-stage infections, L. (L.) infantum was present in all midgut regions, showing typical suprapylarian type of development, whereas L. (V.) braziliensis was concentrated in the hindgut and the abdominal midgut (peripylarian development). We concluded that both Lu. migonei and Lu. longipalpis are equally susceptible vectors for L. (L.) infantum, in laboratory colonies. In relation to L. (V.) braziliensis, Lu. migonei appears to be more susceptible to this parasite than Lu. longipalpis.

As different parasite species may co-circulate in a given endemic area 5 , the significance of co-infections by different Leishmania in sand flies is poorly understood. So far, rather limited number of investigations has been conducted to study the simultaneous development of different Leishmania species in the same sand fly 6,7 . In particular, it would be interesting and important to study simultaneous development of a suprapylarian Leishmania and a peripylarian Viannia.
The present study was designed to fill this gap in knowledge and conducted experimental co-infections to compare the development of L. (L.) infantum and L. (V.) braziliensis in Lutzomyia longipalpis, a known permissive vector 8,9 , and Lutzomyia migonei, which is susceptible to the development of L. (V.) braziliensis 10 and to different strains of L. (L.) infantum 11 . Parasite labelling by different fluorescein proteins enabled us to study interspecific competition during co-infections.
On day 5 PI, L. (L.) infantum infection rate was still high (Fig. 1a), parasites colonized both parts of the midgut (abdominal and thoracic midgut) and, in more than 20% of female sand flies, also the stomodeal valve (Fig. 1b). On the other hand, L. (V.) braziliensis infection rate was lower (50%) and parasites colonized only the midgut and the hindgut. Differences in infection rates were non-significant (X 2 = 4.52, df = 3, P = 0.21), whereas differences in the location of the parasites were significant (X 2 = 6.94, df = 1, P < 0.01). Similar differences between L. (L.) infantum and L. (V.) braziliensis were also observed in parasite loads and their localization during coinfections (Fig. 1b).
Co-infection did not alter the dynamics of infection of the individual parasite species and did not favour or harm any of the parasite species. In co-infections L. (L.) infantum produced high infection rates (Fig. 1a) and colonized the stomodeal valve in high numbers ( Fig. 2D-F). In contrast, L. (V.) braziliensis infections were limited to the midgut and hindgut and some co-infected females became negative for L. (V.) braziliensis (Fig. 1b). 2 PI, infection rates were high in all parasite-vector combination (83%) (Fig. 1c), with parasites present in the endoperitrophic space (Fig. 1d).
On day 8, most of the females of Lu. longipalpis infected with L. (L.) infantum had a heavy infection rate (75%), whereas in L. (V.) braziliensis parasites were less numerous and the infection rate (66%) was significantly lower (X 2 = 38.09, df = 2, P < 0.01) ( Fig. 2A-C). L. (L.) infantum parasites frequently colonized the stomodeal valve while L. (V.) braziliensis infections were mostly limited to in the midgut and pylorus region of the hindgut (Fig. 1d). The same differences in parasite localization were observed in coinfections.
Co-infection did not alter the dynamics of infection of the individual parasite species and did not favour or harm any of the parasite species and the same differences in parasite localization were observed in coinfections.
On day 8 PI, differences between Leishmania species were more pronounced: in Lu. migonei and Lu. longipalpis infected by L. (L.) infantum the majority of parasites were present as elongated nectomonads, whereas in Lu. migonei and Lu. longipalpis infected by L. (V.) braziliensis the prevailing forms were short nectomonads (Fig. 3). Metacyclic forms were found in all parasite-vector combinations, the highest proportion (17.5%) were found in Lu. migonei infected by L. (V.) braziliensis ( Table 1).

Discussion
We evaluated the developmental patterns of two Leishmania spp. in two New World phlebotomine sand fly species, using both single infections and co-infections. Differences found between L. (L.) infantum and L. (V.) braziliensis were more pronounced than the differences between Lutzomyia species. In particular, L. (L.) infantum produced higher infection rates and grew more vigorously, as compared to L. (V.) braziliensis, in both Lu. longipalpis and Lu. migonei. Both sand fly species tested were similarly susceptible to infection by L. (L.) infantum, which confirms previous findings 11 .
Sand fly vectors have been classified into two categories, specific vectors and permissive vectors, based on their ability to support late-stage development of different Leishmania species 8,9 . Specific vectors, like Phlebotomus papatasi support development of a single parasite species or two closely related parasite species 6,12 In contrast, permissive vectors support experimental infections by a broad range of Leishmania spp. The most important example of a permissive vector is Lu. longipalpis, which was involved in the establishment of L. (L.) infantum in Latin America 9 and is proven vector of this parasite in many countries, from Costa Rica to northern Argentina 5,13 . Here we confirmed that Lu. migonei should be also considered as the permissive vector, as females of this species support the development of all Leishmania species tested so far, namely Leishmania (Leishmania) amazonensis, L. (V.) braziliensis and L. (L.) infantum 10,11 .
Nevertheless, our detailed comparative study revealed that Lu. migonei was more susceptible to L. (V.) braziliensis than Lu. longipalpis. Previously, several sand fly species were described as vectors of L. (V.) braziliensis 14   In single infections and co-infections, L. (L.) infantum developed more rapidly in both sand fly species than L. braziliensis. This correlates with growth curves we observed in vitro: during initial three days both species grew similarly, but then, from day 3 onwards, L. (L.) infantum grew significantly faster. These results suggest that development in the sand fly is affected not only by the susceptibility of the vectors, but also by the growth characteristics of the studied strains. In late-stage infections, L. (L.) infantum was present in all midgut regions, the cardia and stomodeal valve in both sand fly species from day 5 PI, showing typical suprapylarian type of development. In contrast, L. (V.) braziliensis was concentrated in the hindgut and the abdominal midgut (peripylarian development). The same happens in the co infections, demonstrating that the two parasites conclude their development and do not compete with each other. Previous study with Lu. migonei and two Leishmania species was done using single infections only, but observations by Nieves and Pimenta 10 were similar to our results: suprapylarian L. (L.) amazonensis grew faster than peripylarian L. (V.) braziliensis.This interspecific difference appears to be strain-independent as almost identical patterns of development were described for the L. (V.) braziliensis strain m2903 10 and for two different L. (L.) infantum strains M4192 and CUK3 in Lu. migonei 11 .
From an epidemiological point of view, it is very important to carry out studies, assessing the development of two species of parasites that cause different forms of leishmaniasis, especially when they coexist in endemic regions 18 . Both Leishmania species completed the life cycle, producing infective forms in both sand fly species studied. We conclude that both Lu. migonei and Lu. longipalpis are equally susceptible vectors for L. (L.) infantum, in laboratory colonies. In relation to L. (V.) braziliensis, Lu. migonei appears to be more susceptible to this parasite than Lu. longipalpis.

Material and Methods
Sand fly colonies and Leishmania strains. Established laboratory colonies of Lu. longipalpis (from Jacobina, Brazil) and Lu. migonei (from Baturité, Brazil) were used and maintained under standard conditions, as previously described 19 . A fluorescent strains of L. (V.) braziliensis (XB29 marked with GFP) and L. (L.) infantum (OGVL marked with mCherry) by stable integration of mCherry into the 18 S rRNA locus as previously described 20 . We have used parasites within less than 10 passages in vitro and were maintained at 23 °C on Medium www.nature.com/scientificreports www.nature.com/scientificreports/ 199 (Sigma-Aldrich, USA) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, USA), 1% BME vitamins (Sigma-Aldrich), 2% human urine and 250 μg/ml amikin (Bristol-Myers Squibb, USA).
For study of growth curves in-vitro, two initial doses were used: 10 4 promastigotes/ml and 10 5 promastigotes/ ml. The concentration of parasites was analyzed daily for 7 days, using counting in Burker chamber. The experiments were repeated twice.
Experimental infections of sand flies. Female sand flies (3-6 days old) of both species were fed through a chick-skin membrane on heat-inactivated rabbit blood containing 10 6 promastigotes/ml for single infections and 5 × 10 5 of each species for co-infections (half a dose used in single infections). Three groups of females of Lu. migonei were studied, the first infected with L. (V.) braziliensis (XB29 marked with GFP), the second with L. (L.) infantum (OGVL marked with mCherry) and the third co-infected with both Leishmania species. The same procedure was performed with L. longipalpis. Engorged females were separated and maintained in the same conditions as the colony and dissected on days 2, 5 and 8 post-infection (PI). Individual guts were placed into a drop of saline and analyzed by fluorescence microscopy for the localization, infection intensity and morphology of Leishmania infections. Parasite loads were graded according to Myskova et al. 21 as light (<100 parasites per gut), moderate (100 to 1000 parasites per gut) and heavy (>1000 parasites per gut). The experiments were repeated four times. infantum and L. (V.) braziliensis, alone or in combination (i.e., co-infected) were prepared on days 5 and 8 PI, being fixed with methanol and stained with Giemsa. Stained smears were then examined under a light microscope with an oil-immersion objective and photographed with an Olympus D70 camera (Olympus, Hong-Kong, China). Body length, body width and flagellar length of 200 randomly selected promastigotes from four females/ smears were measured for each sand fly species and time intervals using Image-J 1.x software. Promastigote forms were distinguished according to Walters et al. 22 and Sadlova et al. 23 . L. (L.) infantum, developmental forms were identified as follows: (i) elongated nectomonads (body length ≥14 μm) (ii) short nectomonads (body length <14 μm and flagellar length ≤2 times body length); (iii) metacyclic promastigotes (body length <14 μm and flagellar length ≥2 times body length); and (iv) haptomonads (flagellum 0-3 µm, present during the late stage infections in cardia). L. (V.) braziliensis, developmental forms were identified as follows: (i) elongated nectomonads (body length ≥13 μm); (ii) short nectomonads (body length <13 μm and flagellar length ≤ 2 times body length); (iii) metacyclic promastigotes (body length <13 μm and flagellar length ≥2 times body length); and (iv) haptomonads (flagellar length ≤ body length, present in hind gut and cardia).