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Adaptation to different nutritional environments is essential for life cycle completion by all Trypanosoma brucei sub-species. In the tsetse fly vector, L-proline is among the most abundant amino acids and is mainly used by the fly for lactation and to fuel flight muscle. The procyclic (insect) stage of T. b. brucei uses L-proline as its main carbon source, relying on an efficient catabolic pathway to convert it to glutamate, and then to succinate, acetate and alanine as the main secreted end products. Here we investigated the essentiality of an undisrupted proline catabolic pathway in T. b. brucei by studying mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase (TbP5CDH), which catalyzes the irreversible conversion of gamma-glutamate semialdehyde (γGS) into L-glutamate and NADH. In addition, we provided evidence for the absence of a functional proline biosynthetic pathway. TbP5CDH expression is developmentally regulated in the insect stages of the parasite, but absent in bloodstream forms grown in vitro. RNAi down-regulation of TbP5CDH severely affected the growth of procyclic trypanosomes in vitro in the absence of glucose, and altered the metabolic flux when proline was the sole carbon source. Furthermore, TbP5CDH knocked-down cells exhibited alterations in the mitochondrial inner membrane potential (ΔΨm), respiratory control ratio and ATP production. Also, changes in the proline-glutamate oxidative capacity slightly affected the surface expression of the major surface glycoprotein EP-procyclin. In the tsetse, TbP5CDH knocked-down cells were impaired and thus unable to colonize the fly’s midgut, probably due to the lack of glucose between bloodmeals. Altogether, our data show that the regulated expression of the proline metabolism pathway in T. b. brucei allows this parasite to adapt to the nutritional environment of the tsetse midgut., Author Summary Bloodsucking insects play a major role in the transmission of pathogens that cause major tropical diseases. Their capacity to transmit these diseases is directly associated with the availability and turnover of energy sources. Proline is the main readily-mobilizable fuel of the tsetse fly, which is the vector of sub-species of Trypanosoma brucei parasites that cause human sleeping sickness and are partly responsible for animal trypanosomiasis (Nagana disease) in sub-Saharan Africa. Once trypanosomes are ingested from an infected host by the tsetse, the parasites encounter an environment that is poor in glucose (as it is rapidly metabolized by the fly) but rich in proline, which then becomes the main carbon source once the parasite differentiates into the first insect (procyclic) stage. In this work, we provide evidence on the essentiality of T. b. brucei proline catabolism for procyclic survival within the tsetse’s digestive tract, as this organism is unable to synthesize this amino acid and strictly depends on the proline provided by the fly. We also show that parasites deficient in TbP5CDH, a mitochondrial enzyme involved in the proline degradative pathway, failed to proliferate in vitro, showed a diminished respiratory capacity, and showed compromised maintenance of energy levels and metabolic flux when proline was offered as the main carbon source. Thus, the integrity of the trypanosome proline degradation pathway is needed to maintain essential functions related to parasite bioenergetics, replication and infectivity within the insect host. Our observations answer a long-standing question on the role of parasite proline metabolism in tsetse-trypanosome interplay.

Enzymes of the glutathione S-transferase (GST) family play critical roles in detoxification of xenobiotics across many taxa. While GSTs are ubiquitous both in animals and plants, the GST epsilon class (GSTE) is insect-specific and has been associated with resistance to chemical insecticides. While both Aedes aegypti and Anopheles gambiae GSTE clusters consist of eight members, only four putative orthologs are identifiable between the species, suggesting independent expansions of the class in each lineage. We used a primer walking approach, sequencing almost the entire cluster from three Anopheles species (An. stephensi, An. funestus (both Cellia subgenus) and An. plumbeus (Anopheles subgenus)) and compared the sequences to putative orthologs in An. gambiae (Cellia) in an attempt to trace the evolution of the cluster within the subfamily Anophelinae. Furthermore, we measured transcript levels from the identified GSTE loci by real time reverse\ud transcription PCR to determine if all genes were similarly transcribed at different life stages. Among the species investigated,gene order and orientation were similar with three exceptions: (i) GSTE1 was absent in An. plumbeus; (ii) GSTE2 is duplicated in An. plumbeus and (iii) an additional transcriptionally active pseudogene (yAsGSTE2) was found in An. stephensi. Further statistical analysis and protein modelling gave evidence for positive selection on codons of the catalytic site in GSTE5 albeit its origin seems to predate the introduction of chemical insecticides. Gene expression profiles revealed differences in expression pattern among genes at different life stages. With the exception of GSTE1, yAsGSTE2 and GSTE2b, all Anopheles species studied share orthologs and hence we assume that GSTE expansion generally predates radiation into subgenera,\ud though the presence of GSTE1 may also suggest a recent duplication event in the Old World Cellia subgenus, instead of a secondary loss. The modifications of the catalytic site within GSTE5 may represent adaptations to new habitats.

Additional file 2: Figure S1. Scheme representing the current leishmaniasis treatment policy in KSA. Figure S2. Leishmania spp. identification in Central Region by PCR-RFLP analysis of parasite ITS1 region. Lane Lt: L. tropica positive control; Lane Lm: L. major positive control; Lanes 1–10: different examples of Leishmania isolates from Rass, Dwadmi and Muzahmyyah. Figure S3. Leishmania spp identification in Al Madinah Province by PCR-RFLP analysis of parasite ITS1 region. Lanes 1–4: different examples of Leishmania isolates from Aljadaida and Sulailah, Al Madinah Province; Lanes 1 and 2: L. tropica samples; Lanes 3 and 4: L. major samples; Lane Lm: L. major positive control; Lane Lt: L. tropica positive control. Figure S4. Leishmania spp. identification in Al Ahsa Region by PCR-RFLP analysis of parasite ITS1 region. Lanes 1–13: different examples of Leishmania isolates from Al Ahsa Region; Lane Lt: L. tropica positive control; Lane Lm: L. major positive control. Figure S5. Leishmania spp identification in Asir Region by PCR-RFLP analysis of parasite ITS1 region. Lanes 1–5: different examples of Leishmania isolates from Asir Province. Figure S6. Distribution of Leishmania species (a) and patient response to anti-leishmanial treatment (b) within the Eastern region of Saudi Arabia. The map was created using software ArcGIS 10 (ESRI, Redlands, CA). Figure S7. Distribution of Leishmania species (a) and patient response to anti-leishmanial treatment (b) within the Northwest region of Saudi Arabia. The map was created using software ArcGIS 10 (ESRI, Redlands, CA). Figure S8. Distribution of Leishmania species (a) and patient response to anti-leishmanial treatment (b) within the southwest region of Saudi Arabia. The map was created using software ArcGIS 10 (ESRI, Redlands, CA).

Additional file 2: Figure S1. Scheme representing the current leishmaniasis treatment policy in KSA. Figure S2. Leishmania spp. identification in Central Region by PCR-RFLP analysis of parasite ITS1 region. Lane Lt: L. tropica positive control; Lane Lm: L. major positive control; Lanes 1–10: different examples of Leishmania isolates from Rass, Dwadmi and Muzahmyyah. Figure S3. Leishmania spp identification in Al Madinah Province by PCR-RFLP analysis of parasite ITS1 region. Lanes 1–4: different examples of Leishmania isolates from Aljadaida and Sulailah, Al Madinah Province; Lanes 1 and 2: L. tropica samples; Lanes 3 and 4: L. major samples; Lane Lm: L. major positive control; Lane Lt: L. tropica positive control. Figure S4. Leishmania spp. identification in Al Ahsa Region by PCR-RFLP analysis of parasite ITS1 region. Lanes 1–13: different examples of Leishmania isolates from Al Ahsa Region; Lane Lt: L. tropica positive control; Lane Lm: L. major positive control. Figure S5. Leishmania spp identification in Asir Region by PCR-RFLP analysis of parasite ITS1 region. Lanes 1–5: different examples of Leishmania isolates from Asir Province. Figure S6. Distribution of Leishmania species (a) and patient response to anti-leishmanial treatment (b) within the Eastern region of Saudi Arabia. The map was created using software ArcGIS 10 (ESRI, Redlands, CA). Figure S7. Distribution of Leishmania species (a) and patient response to anti-leishmanial treatment (b) within the Northwest region of Saudi Arabia. The map was created using software ArcGIS 10 (ESRI, Redlands, CA). Figure S8. Distribution of Leishmania species (a) and patient response to anti-leishmanial treatment (b) within the southwest region of Saudi Arabia. The map was created using software ArcGIS 10 (ESRI, Redlands, CA).

To keep ahead of the evolution of resistance to insecticides in mosquitoes, national malaria control programmes must make use of a range of insecticides, both old and new, while monitoring resistance mechanisms. Knowledge of the mechanisms of resistance remains limited in Anopheles arabiensis , which in many parts of Africa is of increasing importance because it is apparently less susceptible to many indoor control interventions. Furthermore, comparatively little is known in general about resistance to non-pyrethroid insecticides such as pirimiphos-methyl (PM), which are crucial for effective control in the context of resistance to pyrethroids. We performed a genome-wide association study to determine the molecular mechanisms of resistance to deltamethrin (commonly used in bednets) and PM, in An. arabiensis from two regions in Tanzania. Genomic regions of positive selection in these populations were largely driven by copy number variants (CNVs) in gene families involved in resistance to these two insecticides. We found evidence of a new gene cluster involved in resistance to PM, identifying a strong selective sweep tied to a CNV in the Coeae2g-Coeae6g cluster of carboxylesterase genes. Using complementary data from An. coluzzii in Ghana, we show that copy number at this locus is significantly associated with PM resistance. Similarly, for deltamethrin, resistance was strongly associated with a novel CNV allele in the Cyp6aa / Cyp6p cluster. Against this background of metabolic resistance, target site resistance was very rare or absent for both insecticides. Mutations in the pyrethroid target site Vgsc were at very low frequency in Tanzania, yet combining these samples with three An. arabiensis individuals from West Africa revealed a startling diversity of evolutionary origins of target site resistance, with up to 5 independent origins of Vgsc -995 mutations found within just 8 haplotypes. Thus, despite having been first recorded over 10 years ago, Vgsc resistance mutations in Tanzanian An. arabiensis have remained at stable low frequencies. Overall, our results provide a new copy number marker for monitoring resistance to PM in malaria mosquitoes, and reveal the complex picture of resistance patterns in An. arabiensis .