Effect of commensal microorganisms on Leishmania infection of Lutzomyia longipalpis

A wide range of microbial phylotypes have been found associated with adult phlebotomine sand flies collected in the field [4–7, 32] and the percentage of field caught females found to contain a Leishmania population in their gut were usually below 1% [33–35]. Thus Leishmania is only one of many microorganisms vying to occupy the sand fly gut. In the first part of the study we assessed the effect of the interaction between gut microorganisms with Leishmania on subsequent successful colonisation. We used a yeast, Pseudozyma sp. and a bacterium Asaia sp. isolated from the gut of female sand flies collected in a region endemic for visceral leishmaniasis and also O. intermedium, present in our sand fly colony (and other colonies [8]). The α-proteobacterium Asaia used in these experiments was the first of this genus to be isolated from a New World sand fly; in this case a female Lu. longipalpis from a chicken house in Teresina (Piauí-Brazil). There is one record of Asaia being present in a female Phlebotomus sergentii[36]. Asaia were previously isolated from anopheline mosquitoes and are highly prevalent and abundant in their midgut microbiota [37, 38]. It is interesting that both Asaia and the yeast-like fungus Pseudozyma are associated with plants and have osmophilic properties [39, 40] and may have been acquired by the female during plant feeding for sugar-rich food.

To investigate the microbe-Leishmania interaction in vivo, female Lu. longipalpis were given a yeast or a bacterial feed 4 days prior to being membrane-fed with Leishmania mexicana in a bloodmeal. The impact upon the Leishmania population was evaluated by estimating the number of Leishmania promastigotes inside the sand fly gut 72 h after the bloodmeal and the number of sand flies infected were compared to control flies (Figure 1). Prior colonisation by Asaia and O. intermedium significantly reduced the size of the Leishmania populations within the sand flies (Figure 1A) and with O. intermedium, the sand fly infection rates were also reduced (Figure 1B). We also investigated the effect of pre-feeding a combination of two strains of microorganisms, Pseudozyma with Asaia, and there was a significant reduction both in the size of the Leishmania populations and in the number of sand flies containing a Leishmania infection after pre-feeding with the mixed culture (Figure 1A and 1B). This result is consistent with predictions of community ecology theory applied to insect gut microbiota [41] that suggest increasing species diversity within the sand fly gut, by feeding two rather than one species, would lead to a community more resistant to invasion by the Leishmania. It is also possible that the other gut bacteria already present, albeit at low levels (Day 3 onwards: Additional file 1: Figure S2), may have contributed to the colonisation resistance seen towards the Leishmania.

Figure 1 Impact of pre-feeding bacteria and yeast on the subsequent Leishmania population within the gut of female Lu. longipalpis . (A) Leishmania promastigote population estimated within the midgut of Lu. longipalpis after feeding with Pseudozyma sp; Asaia sp. or O. intermedium; 2 - 4 × 107 CFU mL-1. Circles represent individual parasite counts in individual sand fly midguts from 3 independent experiments for each microbial species. *Kruskal-Wallis: P ≤ 0.0001. Mann-Whitney U test: P ≤ 0.007. (B) Percentage of female flies infected with Leishmania. Control group were fed on 7% w/v sucrose before being fed with Leishmania. *Fisher’s Exact Test P ≤ 0.0001. Full size image

The effects were further studied by comparing two O. intermedium concentrations of 106 and 107 cfu mL-1 fed to female Lu. longipalpis 4 days prior to being fed with L. mexicana. The results showed that there was no concentration dependent effect as the Leishmania promastigote population was not reduced when flies were membrane-fed with O. intermedium at 107 CFU mL-1, in comparison with flies fed the lower concentration of 106 CFU mL-1 (Figure 2A). A sand fly is estimated to ingest less than 0.8 μl of fluid in a meal [19], this suggests that an initial dose of < 800 bacteria was sufficient to reduce the subsequent Leishmania population in sand flies. We also showed that the Ochrobactrum needed to be alive to exert these effects, as the Leishmania infection rates observed in sand flies fed with heat-inactivated bacteria at 107 CFU mL-1 were not significantly different from the controls (Figure 2B). This result together with the observation that only bacterial or yeast cells, and not supernatant, showed growth limiting effects towards Leishmania in vitro (Additional file 2: Figure S1A & B) suggest that microbial interference in Leishmania development is likely to occur only when the live cells are present.

Figure 2 Effect of O. intermedium concentration and heat inactivation on the percentage of female Lu. Longipalpis containing a Leishmania infection. (A) Two concentrations of O. intermedium of 106 (OD 0.02) and 107 CFU mL-1 (OD 0.2) were used to feed groups of sand flies prior to Leishmania infection. Control group were fed on 7% sucrose only before infections. Three independent experiments were carried out. Fisher’s Exact Test:* P ≤ 0.0001. (B) Effect of feeding heat inactivated (HI) O. intermedium (107 CFU mL-1) on subsequent Leishmania percentage infection of females. Infection rates from individual sand fly midguts from 3 independent experiments. Fisher’s Exact Test: *P ≤ 0.0001. Full size image

The sand flies used in the colonisation study were reared in an aseptic environment with minimal microbial contamination but were not “germ free”. The aseptically prepared adults were assessed using QPCR for the bacterial 16S rDNA gene and the results indicated a low level of bacteria associated with these insects that did not change significantly after bloodfeeding (Additional file 1: Figure S2). Newly emerged adults were associated with significantly larger amounts of bacterial DNA that were not evident in 3 day old adults. We suggest that the bacterial DNA was associated with the meconium [42] that would have contained bacteria (dead or alive) as remnants of the larval gut. The meconium is voided from the adult gut within hours of the adult emerging from the pupae. Therefore, the sand flies may have contained gut bacteria carried over from the larval stages after pupation or from environmental contamination. Antibiotic treatment to eliminate the gut bacteria was not used as relevant antibiotics would potentially also inhibit Leishmania development. Germ free insects were not used as the immune response of these insects may differ markedly from a conventionally reared insect [43] and the immune response may form a vital function as part of the interaction between sand fly host, Leishmania and bacteria or yeast [23, 44].

The majority of the studies describing the effects of gut microbiota on parasite prevalence and development within medically important insect vectors have been done with mosquitoes [45], tsetse flies [13] and triatomine bugs [12]. Plasmodium is only found within the mosquito midgut for a limited period of time in comparison to Leishmania in the gut of their insect vector. However, a positive correlation was observed between the presence of a midgut microbiota and inhibition of Plasmodium development within the mosquito [43]. This inhibition was variously attributed to direct competition [43], active production of ROS by indigenous mosquito microbiota [46] or a consequence of activation of the basal immunity generated by mosquito gut bacteria [47]. Although very dominant in anopheline microbiota, there is no evidence that the symbiotic Asaia induce colonisation resistance in mosquitoes [38].

Our previous studies indicate that immune activation of the sand fly can lead to loss of Leishmania infection [23] and that although Leishmania do not appear to activate a ROS response, bacteria in the gut do cause ROS activation [44]. Direct production of antimicrobial compounds by bacteria, as found in other insects [48] cannot be discounted. The mechanisms of colonisation resistance in sand flies are therefore likely to be multifaceted; including direct bacterial-mediated lysis, competition for binding sites and nutrients or indirect via immune priming of the sand fly host. An intriguing parallel situation is demonstrated in plant systems with the epiphytic Pseudozyma and plant pathogen Botrytis cinerea[49]. Pseudozyma aphidis secreted extracellular metabolites not only inhibit the pathogen but also primes the plant immune system to invoke a local and systemic immune response towards the pathogen.

Role of Leishmania in protecting Lutzomyia longipalpis from an insect pathogen

The final part of the investigation addressed the hypothesis that Leishmania are beneficial to the sand fly host. There are many microbial species encountered by the sand fly vector that are potentially pathogenic to the insect. We investigated circumstances in which the Leishmania may prevent the development of a sand fly pathogen. The colonisation experiments were repeated but the feeding regime was reversed; we fed bacteria to sand flies already colonised with Leishmania. We used the insect bacterial pathogen, Serratia marcescens as it is known to be associated with wild Lu. longipalpis and is also lethal to Leishmania in vitro[7, 25]. Preliminary in vitro experiments confirmed that both the cells of S. marcescens and the spent culture media incubated with our strain of L. mexicana led to suppression of parasite growth (Figure 3).

Figure 3 Effect of S. marcescens on growth of L. mexicana in vitro . (A) In vitro 24 h incubation of Serratia bacterial cells (107 CFU mL-1) or (B) filtered spent medium from Serratia culture with L. mexicana (3 × 106 promastigotes mL-1). Results are based on triplicate samples repeated three times and bar charts represent mean ± SEM. *P ≤ 0.009. **P ≤ 0.0001 (Mann-Whitney U test). Full size image

Flies that were blood fed and then subjected to a daily feed of Serratia in a sugar meal (Figure 4A) succumbed to the bacterial infection and only 11% survived after 6 days. In contrast, sand fly survival was significantly higher (56% after 6 days; Figure 4A) when fed a blood meal containing L. mexicana amastigotes and then subjected to a daily feed of Serratia in a sugar meal. The presence of Leishmania in the gut enhanced the survival of Serratia-challenged sand flies in comparison with those not infected with Leishmania. Remarkably, the population of Leishmania in these sand flies was not significantly different to those in a further set of control flies that were not fed Serratia (Figure 4B, Additional file 3: Figure S3). Survival of sand flies infected with Leishmania but not challenged with Serratia was no different to that of control sand flies (Figure 4A).

Figure 4 Effect of Leishmania infection on sand fly survival after oral challenge with Serratia marcescens . (A) Survival of female Lu. longipalpis containing Leishmania after oral challenge with Serratia marcescens in sucrose (diamond) in comparison with insects fed with a bloodmeal containing Serratia (square), Leishmania (triangle), or blood followed by sucrose (circle). **P ≤ 0.0001; Chi-square 96.987 (Log Rank-Mantel Cox). (B) Scatter plot showing Leishmania promastigote population, at day 3, within individual sand fly midguts after subsequent feeding with 20% w/v sucrose or a Serratia marcescens suspension (5.7 × 107 CFU mL-1) via cotton wool. Full size image

When an insect vector containing a medically important parasite is exposed to a pathogen of the vector it threatens the successful transmission of the parasite. In our experiments when the Leishmania infected sand flies were subsequently fed with Serratia, the Leishmania population was similar to that of the control insects. This suggests that the association between Leishmania and its insect vector promote both survival of the insect and the flagellate population. Additionally there was no difference in the survival of flies with Leishmania infection with or without the Serratia challenge during the period of the experiment.

There was a significant reduction in the population of naturally-occurring sand fly gut bacteria sampled at 3 days (Figure 5A) in Leishmania infected sand flies that had been exposed to a daily Serratia oral challenge. However, there were no differences in Serratia populations in surviving flies infected with Leishmania in comparison with the flies without Leishmania (Figure 5B). The decrease in gut microbiota is consistent with the idea that colonisation resistance was generated by Leishmania towards indigenous bacteria within the sand fly. It should be noted that sampling of blood fed sand flies without Leishmania may have resulted in a lower than expected population of Serratia since flies with higher Serratia doses probably died before the time of sampling.

Figure 5 Estimate of naturally-occurring sand fly gut bacteria. (A) and Serratia marcescens (B) in Leishmania infected flies after oral challenge with Serratia. Estimated as CFUs present in individual Lu. longipalpis midguts either uninfected (Blood + Serratia) or infected with L. mexicana (Leish + Serratia) at 3 days after daily oral challenge with Serratia marcescens via cotton wool (5.7 × 107Serratia CFUmL-1, resuspended with sterile 20% w/v sucrose solution). Asterisk represents statistical difference using Mann-Whitney U test (P ≤ 0.03) of at least two independent experiments. Full size image

These experiments highlighted one circumstance in which the association between Leishmania and sand flies may be mutually beneficial. Increased protection from insect pathogens will extend the lifespan of sand flies. But does the potential benefit of the Leishmania help to offset the known fitness costs [18] of a Leishmania population in the sand fly gut? This is a significant question since even a few days life extension in a disease endemic area may greatly promote Leishmania transmission and contribute to the successful spread of the human disease. An increased disease resistance conferred on the insect by Leishmania could be an important evolutionary pressure for the maintenance of the Leishmania-vector association. Sand flies more resistant to Leishmania infection may be more exposed to enteric bacterial entomopathogens. In this case maintenance of a small fraction of Leishmania-susceptible flies in the vector species may ensure insect survival within a population that is succumbing to an entomopathogen.

A further implication of the protective effect of Leishmania is that implementing a biological control campaign against insect vectors using insect pathogens may have unwarranted effects. Sand flies not carrying Leishmania may succumb more rapidly to the biological control agent and this would lead to the development of a wild sand fly population containing an increased proportion of the surviving flies carrying the human disease agent. Any new forms of control aimed at insect vectors of human disease need to consider the total macro- and micro- ecology of the relationship between the insect and the human parasite.