The effect of the sugar metabolism on Leishmania infantum promastigotes inside the gut of Lutzomyia longipalpis: A sweet relationship?

It is well-known that Leishmania parasites can alter the behavior of the sand fly vector in order to increase their transmission potential. However, little is known about the contribution of the infecting host’s blood composition on subsequent sand fly infection and survival. This study focused on the host’s glucose metabolism and the insulin/insulin-like growth factor 1 (IGF-1) pathway as both metabolic processes are known to impact vector-parasite interactions of other protozoa and insect species. The focus of this study was inspired by the observation that the glycemic levels in the blood of infected Syrian golden hamsters inversely correlated to splenic and hepatic parasite burdens. To evaluate the biological impact of these findings on further transmission, Lutzomyia longipalpis sand flies were infected with blood that was artificially supplemented with different physiological concentrations of several monosaccharides, insulin or IGF-1. Normoglycemic levels resulted in transiently higher parasite loads and faster appearance of metacyclics, whereas higher carbohydrate and insulin/IGF-1 levels favored sand fly survival. Although the recorded effects were modest or transient of nature, these observations support the concept that the host blood biochemistry may affect Leishmania transmission and sand fly longevity.


Author summary
Past research on the interaction between the Leishmania parasite and the sand fly vector has revealed that Leishmania is capable of changing vector behavior to favor transmission of parasites in the environment. Little is known about the impact of host blood composition on parasite development inside the vector and on vector survival. Here, we showed that parasite burdens in the spleen and the liver inversely correlated to the serum blood glucose levels of infected animals, which triggered us to further investigate the effect of blood monosaccharides, insulin and insulin-like growth factor 1 (IGF-1) on sand fly infection and survival. We demonstrated that normal serum glucose levels in the initial parasitized blood meal resulted in transiently higher parasite loads and a faster appearance of infectious parasites, whereas higher sugar and insulin/IGF-1 levels favored sand fly

Introduction
Leishmaniasis is a neglected tropical disease caused by the protozoon Leishmania parasite.
Depending on the infecting parasite species the disease manifests itself as cutaneous leishmaniasis (causing skin lesions), mucocutaneous leishmaniasis (destroying cartilage and soft tissue of the nose, pharynx and palate) or visceral leishmaniasis (affecting spleen, liver and bone marrow). This family of related diseases is prevalent in over 98 countries and, although a lot of infections remain asymptomatic, the visceral disease still causes ten thousands of deaths each year. Disease control mainly relies on prompt and accurate diagnosis and treatment and, as the parasite is transmitted through the bites of female sand flies of the genera Phlebotomus and Lutzomyia, on vector control. Infection with Leishmania parasites is known to provoke several changes in the fly's feeding behaviour, hereby increasing the insect's biting persistence and its tendency to feed on multiple hosts, thus boosting parasite transmission [1,2]. For Plasmodium, a link has been demonstrated between the presence of human hormones, cytokines, and growth factors that are ingested with the parasitized blood meal and the infection dynamics in the mosquito and transmission potential to other hosts [3]. First of all, low glucose levels are associated with impaired parasite growth and proliferation in vitro [4]. Secondly, mice having type 2 diabetes, which is associated with elevated steady-state blood glucose levels, show an increased transmission potential to mosquitos although no significant differences in circulating gametocyte levels were noted [5]. The higher glucose levels have shown to increase the attractiveness of the host for vector feeding [6] via the production of several mosquito-attracting volatiles [7,8]. Thirdly, Plasmodium infections are often associated with hypoglycaemia and hyperinsulinemia. By altering the insulin/insulin-like growth factor (IGF) signalling (IIS) pathway inside the vector gut, human insulin levels not only increase mosquito susceptibility to Plasmodium infection, but also alter vector immunity favouring mosquito survival and parasite transmission potential [9,10].
This highly conserved IIS pathway, which is known to be implicated in Plasmodium-mosquito interactions, also regulates the metabolism, development, lifespan and immunity in other organisms, such as Drosophila. Although homologs of the IIS pathway were also identified in sand flies [11], little research hitherto has focused on Lutzomyia and Phlebotomus spp., leaving a gap in our understanding of the sand fly immune response to Leishmania infection and the effects of the blood composition on subsequent parasite infection and transmission.
Nevertheless, previous research also showed a positive correlation between the glucose availability in the culture medium and in vitro promastigote growth for Leishmania [12]. In addition, decreased glucose concentrations because of malnourishment are well known to decrease immune responses during active leishmaniasis and therefore increase host susceptibility to infection both in experimental animal models and in humans [13][14][15][16][17]. Whether infection can also influence glucose levels in the host and whether prandial glucose levels might impact the infection in the sand fly vector have not been investigated so far. This study therefore aimed to characterize the potential relationship between the host's glucose/insulin metabolism and its effects on Leishmania infection in the sand fly vector and on subsequent parasite transmission.

Ethics statement
The use of laboratory rodents was carried out in strict accordance to all mandatory guidelines (EU directives, including the Revised Directive 2010/63/EU on the Protection of Animals used for Scientific Purposes that came into force on 01/01/2013, and the declaration of Helsinki in its latest version) and was approved by the ethical committee of the University of Antwerp, Belgium (UA-ECD 2020-17 (05-05-2020).

Animals, sand flies and parasites
Female Swiss mice (BW 20-25 g) and Syrian golden hamsters (100-120 g) of six-eight weeks old were purchased from Janvier (France) and kept in quarantine for at least 5 days before infection or blood collection. Food for laboratory rodents and drinking water were available ad libitum.
A Lutzomyia longipalpis sand fly colony was initiated with the kind help of NIH-NIAID (Prof. Shaden Kamhawi and Prof. Jesus Valenzuela) and maintained at the University of Antwerp under standard conditions (26˚C, >75% humidity, in the dark) with provision of a 30% glucose solution ad libitum [18]. For infection and transmission experiments, 3-to 5-day old females from generations 21 to 34 were used.
A recent clinical Leishmania infantum isolate (MHOM/ES/2016/LLM-2346) was kindly provided by Dr. J. Moreno from the World Health Organization Collaborate Centre (WHOCC, Madrid, Spain). Promastigotes were routinely cultured in T25 culture flasks containing 5 mL of HOMEM hemoflagellate medium, supplemented with 10% inactivated fetal bovine serum (iFBS), and were subcultured twice weekly. As parasite virulence is known to rapidly decline upon prolonged in vitro passage, infections were caried out using low-passage promastigotes (<5 passages). To obtain highly virulent ex vivo amastigotes, parasites were also expanded in vivo in hamsters. For this purpose, hamsters were infected intracardially with an infection inoculum containing 2×10 7 parasites/100 μL RPMI-1640. Six to 8 weeks post infection, ex vivo amastigotes could be harvested from the spleen, quantified using the method described by Stauber [19] and suspended at a final concentrations of 2×10 7 amastigotes/100μL in RPMI-1640 to initiate novel infections.

Hamster studies
To evaluate whether parasite burdens in the main target organs (spleen, liver and bone marrow) could be correlated to serum glucose levels, groups of three hamsters were infected intracardially with a 1:10 dilution series of ex vivo amastigotes in RPMI-1640 ranging from 10 8 to 10 4 parasites/hamster. At 6 weeks post infection (appearance of clinical symptoms in the group infected with the highest infection dose) sublingual blood was collected to determine serum glucose levels. Hamsters were then euthanized with CO 2 and spleen, liver and bone marrow were harvested and used to prepare tissue imprints on glass microscopic slides. The slides were fixed with methanol and stained with a 1:5 Giemsa dilution in water, allowing the microscopic determination of parasite tissue burdens [expressed as Leishman Donovan Units for liver and spleen (LDU) and as the average number of amastigotes per macrophage for bone marrow].

Promastigote growth curves
To generate promastigote growth curves, promastigotes were counted using a KOVA counting chamber and seeded in new T25 flasks containing exactly 5 mL promastigote medium at a density of 5×10 5 promastigotes/mL. To evaluate the growth effects caused by the different types of monosaccharides, IGF-1 and insulin, HOMEM medium without glucose or iFBS, but supplemented with 5 g /L Albumax II, was used. This medium was then spiked with the desired concentrations of glucose, [70 mg/dL (low) or 180 mg/dL (high)], fructose [0.005 nM (low) or 0.15 nM (high)], galactose [10 mg/dL (low) or 100 mg/dL(high)], IGF-1 (50 ng/mL) or insulin (100 mIU/L). Cultures seeded in HOMEM containing Albumax II without the additives were included as controls. Cultures were incubated at 25˚C for 10 days. Each 24h, the parasite density in the cultures was counted using a KOVA counting chamber.

Preparation of blood
Blood was collected via intracardial puncture of euthanized, fasted Swiss mice using heparincoated syringes (Becton Dickinson). Upon its collection, blood was kept on ice. It was first centrifuged at 4˚C at 930×g for 10 minutes to separate the cells from the plasma. The cells were washed twice with ice-cold PBS and again centrifuged at 4˚C at 930×g for 10 minutes. The collected plasma was placed at 56˚C for 45-60 minutes to allow inactivation of complement. Afterwards, the plasma was chilled on ice and again mixed with the cells. Finally, 30 μL penicillin/streptomycin was added per mL of blood. Blood glucose levels were evaluated using the OneTouch Verio glucose meter and blood was only used if glucose levels were > 70 mg/dL.
For the experiments with infected blood, 24h-old log phase promastigotes were centrifuged and washed with PBS at 2850×g for 15 minutes at room temperature. After the last washing step, the PBS was removed and the promastigotes were quantified using a KOVA-counting chamber. The blood was infected by adding 5×10 6 promastigotes/mL.

Spiking of blood
The blood was then spiked with the different proteins and sugars at a physiological range between a minimum (low), a medium (med) and a maximum (high) concentration ( Table 1). The medium concentration is chosen to correspond to the normal physiological concentration of a healthy human individual.

Sand fly infections
Sand flies were deprived of sugar 14-16 h before infection. For artificial infections, 200-300 flies were placed in infection cups before infection. These small, plastic cups were covered with  [3,9] an insect mesh through which flies were offered access to sugar or blood meals. The prepared blood was placed into a glass feeder device covered with a chick skin-membrane and placed on the mesh of the infection cup for 2 h, allowing the flies to take a blood meal. Twenty-four hours after infection blood-fed females were isolated and further maintained at 26˚C to evaluate the course of infection over time. To avoid overcrowding and associated premature death, flies were kept with a maximum of 150 females in one infection cup. During the follow-up period of the experiment flies were given access to a 30% sucrose solution ad libitum, to mimic their access to sugary plant secretions in nature.

Evaluation sand fly survival
Sand fly survival was evaluated at days 2, 5, 7 and 9 after infection by counting the number of death flies per condition. Based on the number of dead flies removed from the cages, Kaplan-Meier curves were constructed using Graphpad prism version 6.0.

Evaluation parasite load and metacyclogenesis in sand fly
To evaluate the total parasite load in the fly gut and the rate of metacyclogenesis, sand fly guts were dissected at days 2, 5, 7 and 9 after infection. The gut was macerated in 50 μL PBS with a plastic pestle and the number of parasites inside the gut was microscopically determined using a KOVA counting chamber. In parallel, the percentage of metacyclics was determined microscopically in the same KOVA counting chamber based on parasite morphology and motility as established elsewhere [27,28]. All experimental infections were performed with an independent repeat for each condition. To avoid bias during counting and microscopic evaluation of morphology and motility, groups were randomized by an independent researcher until data analysis.

Statistical analysis
All statistical analyses were performed using SPSS and Graphpad Prism version 6.00 software. Statistical differences between the different experimental sand fly groups were determined by 2-way ANOVA, whereas differences between groups in the Kaplan-Meier survival analyses were determined by Pearson Chi-Square analysis. The potential correlation between serum glucose levels and parasite tissue burdens and transmission were analyzed using a Spearman ranked test. Tests were considered statistically significant if p <0.05.

Monosaccharide levels do not significantly impact promastigote growth in vitro
Despite earlier reports indicating a possible growth-stimulating effect of glucose on in vitro promastigote cultures [12], no growth-enhancing effect was observed under our experimental conditions upon increasing the monosaccharide concentration in the culture medium (S1 Fig). Similarly, the addition of IGF-1 or insulin to the in vitro culture medium also had no significant effect on promastigote growth.

Normal blood sugar levels are most compatible with Leishmania development in the sand fly
When evaluating the effect of the various monosaccharide concentrations on infection inside the vector gut over time (Fig 2), little effects were observed. On day 5 and day 7 of the

Fig 1. Blood glucoses levels in the blood of infected hamsters in relation to the parasite burdens in spleen (A-C), liver (B-D) and bone marrow (E).
Six weeks after infection sublingual blood was collected from hamsters infected with different parasite burdens and the blood glucose concentration was determined. Hamsters were autopsied afterwards and the parasite burdens in the target main organs (liver, spleen and bone marrow) were determined based on microscopic counting of the number of amastigotes on Giemsa-stained tissue imprints. Results were analysed using the nonparametric Spearman correlation analysis (correlation coefficients r indicated in the graphs) and with inclusion of a linear regression. A weak to moderate correlation between both parameters could be found in the spleen and the liver. For the bone marrow both parameters could not be correlated. Results are based on data derived from 3 biological repeats in 15 individual hamsters (5 groups of 3 animals). Each group received a different infection inoculum containing either 10 8 , 10 7 , 10 6 , 10 5 or 10 4 parasites.
https://doi.org/10.1371/journal.pntd.0010293.g001 infection, medium concentrations of glucose, which correspond to the normal physiological concentrations found in healthy human beings, resulted in a slightly higher parasite load in comparison to the hypo-and hyperglycemic conditions. Despite the limited effects of monosaccharide concentrations on gut parasite load, the effect on metacyclogenesis was found more pronounced (Fig 3). Medium concentrations of glucose led to the earlier development of metacyclics in the vector gut, which suggests again that normal physiological glucose concentrations are optimal for transmission. For fructose and galactose, high saccharide concentrations exerted a negative impact on metacyclogenesis.

Increased blood sugar levels enhance sand fly survival
To evaluate whether the addition of glucose to the blood meal also had an impact on sand fly survival, the number of dead sand flies was recorded during the infection experiments and a Kaplan-Meier analysis was performed (Fig 4). This analysis revealed a significant benefit of feeding on the high glucose concentrations for sand fly survival. Flies fed on hypoglycemic blood had the lowest survival rates.

Blood insulin and IGF1 levels do not impact Leishmania development in the sand fly
Spiking the infective blood meal with different physiological concentrations of insulin did not impact parasite load or metacyclogenesis (Fig 5A and 5B). Changing IGF-1 concentrations in the infective blood meal also did not lead to differences in gut establishment and parasite maturation (Fig 5C and 5D). Only on day 7 post infection, parasite levels and percentage metacyclics tend to be highest in the flies fed on blood with normal IGF-1 concentrations (medium). Higher IGF-1 levels seem to rather negatively impact the infection.

High insulin or IGF-1 levels favor sand fly survival
Although the blood insulin concentration has no effect on parasite infection in the sand fly gut (Fig 5A and 5B), medium and high insulin concentrations in the blood meal were able to significantly enhance sand fly survival over the course of the experiment (Fig 6A). When

PLOS NEGLECTED TROPICAL DISEASES
Effect of glucose metabolism on experimental sand fly infections assessing sand fly survival upon blood feeding with different IGF-1 concentrations over time (Fig 6B), IGF-1 initially does not seem to impact vector survival. However, final survival was highest in the group feeding blood with high IGF-1 concentrations (day 9).

Discussion
With the increasing spread of Western diet and lifestyle worldwide, overweight and the socalled "civilization diseases", such as diabetes mellitus type 2, are also rapidly increasing in several developing countries [29]. The emerging co-occurrence of type 2 diabetes mellitus and tropical infectious diseases, however, may have substantial implications as type 2 diabetes mellitus is known to increase the body's susceptibility to common infections [30]. Infections also significantly impact host immunological and physiological parameters. In this study, artificial infection of Syrian golden hamsters with L. infantum parasites revealed a moderate inverse correlation between serum blood glucose levels and tissue burdens in the spleen and liver. This finding prompted us to further evaluate the impact of the host serum composition on sand fly infection and survival. For other infectious diseases, such as malaria, the presence of human hormones, cytokines, and growth factors (such as glucose and insulin) in the infectious blood meal can indeed alter the course of infection, hereby affecting vector physiology and parasite transmission [3].
Carbohydrates are not only very important for sand fly biology, but also for Leishmania development and transmission. Although female sand flies need a blood meal to initiate ovarian development and oviposition, sand flies mainly feed on plant juices and sugary secretions. These ingested sugars are very important for subsequent parasite development and anterior migration in the sand fly gut [31][32][33][34][35][36][37] and are key drivers for metacyclogenesis [12] and the formation of the parasite's LPG coat [37][38][39][40][41], which is altered upon parasite detachment from the fly's midgut [41][42][43][44][45]. Moreover, sand fly survival decreases in the absence of sugars [31].
As saccharides may clearly impact parasite development inside the sand fly vector, their impact on promastigote multiplication was first evaluated in vitro. Under the used  experimental conditions, no effect of the addition of high saccharide concentrations in the culture medium could be observed. When assessing the effect of monosaccharide addition to the infective blood meal, it was therefore not surprising to observe that higher concentrations of monosaccharides did not enhance parasite multiplication. Normal physiological glucose concentrations were compatible with the highest parasite load in the sand fly gut and an increased proportion of metacyclics early in infection (day 5). Our results also show that intake of a sugar-rich blood meal increases the survival of L. longipalpis, a phenomenon that has already been reported for Drosophila [46]. Dietary glucose additionally enhanced immunity against enteric infections with the Gram-negative Vibrio cholerae bacteria in adult Drosophila [47]. Whether this effect in L. longipalpis is also caused by an enhancing effect on insect immunity, remains to be investigated.
Given the differences in the biology and ecology each pathogen-vector interaction, research on phlebotomines is essential to identify parallels or unique features of the Leishmania infection and transmission process. Hosts infected with Plasmodium demonstrate increased blood insulin concentrations, which trigger the insulin/IGF-1 signaling pathway in Anopheles mosquitos, hereby increasing mosquito susceptibility to infection, lifespan and transmission [10,48]. For Leishmania, increased insulin concentrations did not result in increased vector susceptibility to infection. However, high and medium concentrations of insulin seem to extend the lifespan of the vector similar to what was observed for malaria mosquitos [10]. IGF-1 on the other hand, was shown to render mosquitos more resistant against Plasmodium falciparum infections and to increase the mosquito lifespan [3,9]. In line with these findings, high IGF-1 concentrations during infection of L. longipalpis reduced parasite loads and metacyclogenesis at day 7 post infection and an enhanced sand fly survival by the end of the experiment. Highest parasite levels and proportion of metacyclics were observed with normal IGF-1 levels. Although our results corroborate earlier work performed for Plasmodium, the effect of this specific factor on L. infantum infection in L. longipalpis is considerably less pronounced. Possibly, the differences observed between L. longipalpis sand flies, Drosophila fruit flies and Anopheles mosquitos are caused by differences in their immune systems. For Drosophila and Anopheles most immunological pathways involved in symbiosis with gut microbiota and defense against parasitic infections are fairly well characterized [49][50][51][52], with the Toll pathway and immune deficiency (Imd) pathway being important contributors to Drosophila and mosquito immunity. In Drosophilla, recent research even revealed the implication of Imd pathway activation in metabolic regulation (hyperglycemia, depleted fat reserves, reduced survival, reduced size and developmental delays) and immunity (increased susceptibility to infections) [53][54][55][56][57][58]. Past research on two L. longipalpis-derived cell lines, LL-5 and Lulo, had already been suggestive for the potential involvement of both these pathways in innate immune responsiveness against Leishmania [59]. Recent research indeed corroborated the upregalation of both pathways in sand flies upon a blood meal, they were not significantly upregulated in the presence of Leishmania and most likely react to the increase in sand fly midgut bacteria in the presence of blood [60][61][62]. However, the link between parasite survival, gut microbiota and the immune responses mounted by the sand fly definitely needs further investigation.
Collectively, the observations in this study show that host blood biochemical parameters of the glucose metabolism impact sand fly longevity and cause modest, transient effects in Leishmania development in the insect gut. In general, higher physiological carbohydrate and insulin/IGF-1 seem to enhance fly survival whereas normoglycemic conditions transiently favor early development of metacyclics. Although these observations may have a limited epidemiological impact, they support the concept that host blood biochemical parameters may affect Leishmania transmission and sand fly longevity.

S1 Fig. Comparison of in vitro promastigote growth of L. infantum cultured in either medium without additives (ctr) or in media containing low of high levels of glucose (A), fructose (B) or galactose (C) or insulin and IGF-1 (D)
. No significant differences in L. infantum promastigote growth could be observed. The parasite density at each time point of cultivation is the average of at least two independent experiments run in duplicate ± the standard error of the mean. (TIF)