Acta Tropica 122 (2012) 80–86 Contents lists available at SciVerse ScienceDirect Acta Tropica j o ur nal homep age : www.elsevier.com/locate/ actatropica Individual variability of salivary gland proteins in three Phlebotomus species Iva Rohouˇ sová ∗ , Vˇ era Volfová, So ˇ na Nová, Petr Volf Department of Parasitology, Faculty of Science, Charles University, Viniˇ cná 7, 128 43 Prague 2, Czech Republic a r t i c l e i n f o Article history: Received 16 September 2011 Received in revised form 25 November 2011 Accepted 9 December 2011 Available online 16 December 2011 Keywords: Phlebotomus Phlebotomine sand flies Salivary glands Individual variability Leishmania transmission Anti-Leishmania vaccine a b s t r a c t Pooled salivary gland samples are frequently used to ensure the sufficient amount of material for the experiments; however, this could mask an individual variability. Thus, we compared salivary protein profiles in seven colonies of three Phlebotomus species: Phlebotomus sergenti, Phlebotomus perniciosus, and Phlebotomus papatasi. Surprisingly, the individual profiles differed significantly between the colonies as well as between individuals. The highest variability was observed in proteins with molecular masses of 42–46 kDa corresponding to the yellow-related proteins. The phenogram constructed from salivary gland profiles revealed the existence of two main groups in P. sergenti, corresponding well with the geographical origin. The F1 progeny obtained from cross-mating studies between P. sergenti colonies of different geographical origin formed a distinct subgroup within the parental groups. In P. papatasi, several groups of protein profiles were observed with no relationship to the geographical origin. The biological role of salivary proteins variability is discussed. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Sand flies as haematophagous insects must cope with the haemostasis of the vertebrate host. Thus they are armed with a battery of salivary antihaemostatic and immunomodula- tory molecules. While overcoming haemostatic reactions, those molecules can also cause discomfort to the host such as pain, itching, oedema, and hypersensitive reactions. Moreover, salivary molecules may influence pathogens vectored by sand flies, some of them of prime medical and veterinary importance such as Phle- boviruses, Bartonella bacilliformis, and Leishmania (Rutledge and Gupta, 2002). Indeed, sand fly saliva facilitates the establishment of Leishmania infection in the naive host advancing Leishmania multi- plication (Oliveira et al., 2009; Rohousova and Volf, 2006). On the other hand, host pre-exposed to sand fly saliva develop immune reactions reducing the risk of severe leishmaniasis (Belkaid et al., 1998; Kamhawi et al., 2000; Thiakaki et al., 2005) thus vol- unteering for alternative anti-Leishmania vaccine strategy. This vaccine concept was already proven in some laboratory models for single salivary proteins such as SP15 from Phlebotomus pap- Abbreviations: P., Phlebotomus; L., Lutzomyia; UPGMA, Unweighted Pair Group Method with Arithmetic Mean. ∗ Corresponding author at: Department of Parasitology, Faculty of Science, Charles University, Viniˇ cná 7, 128 44 Prague 2, Czech Republic. Tel.: +420 221 951 814; fax: +420 224 919 704. E-mail addresses: rohousova@seznam.cz (I. Rohouˇ sová), veravolf@seznam.cz (V. Volfová), nova.sona@gmail.com (S. Nová), volf@cesnet.cz (P. Volf). atasi (Valenzuela et al., 2001) or LJM19 from Lutzomyia longipalpis (Gomes et al., 2008; Tavares et al., 2011). Various sand fly species differ in salivary proteins/peptides and in their antigenic and pharmacological properties (Anderson et al., 2006; Cerna et al., 2002; Lanzaro et al., 1999; Rohousova et al., 2005; Volf and Rohousova, 2001). Certain degree of variability has also been found between different populations of the same species – e.g. Phlebotomus duboscqi or L. longipalpis – both on mRNA and pro- tein levels (Kato et al., 2006; Lanzaro et al., 1999; Volf et al., 2000). Such variability may have an impact on clinical manifestation of Leishmania infection; in the case of L. longipalpis sibling species, geographically different populations vary in the amount of sali- vary peptide maxadilan thus influencing the outcome of Leishmania infantum chagasi infection (Warburg et al., 1994). The objective of this study was to determine individual vari- ability of sand fly salivary proteins. We analyzed salivary proteins of P. (Phlebotomus) papatasi, Phlebotomus (Larroussius) perniciosus, and Phlebotomus (Paraphlebotomus) sergenti, all being important vectors of Leishmania parasites in the Old World (Killick-Kendrick, 1999). We demonstrated surprisingly high degree of individual variability in salivary proteins and discussed the possible biological consequences of such diversity. 2. Materials and methods 2.1. Sand fly saliva Sand fly colonies were reared in standard conditions as described previously (Volf and Volfova, 2011). P. sergenti colonies 0001-706X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2011.12.004