Review
An insight into the sialome of blood-feeding Nematocera

https://doi.org/10.1016/j.ibmb.2010.08.002Get rights and content

Abstract

Within the Diptera and outside the suborder Brachycera, the blood-feeding habit occurred at least twice, producing the present day sand flies, and the Culicomorpha, including the mosquitoes (Culicidae), black flies (Simulidae), biting midges (Ceratopogonidae) and frog feeding flies (Corethrellidae). Alternatives to this scenario are also discussed. Successful blood-feeding requires adaptations to antagonize the vertebrate’s mechanisms of blood clotting, platelet aggregation, vasoconstriction, pain and itching, which are triggered by tissue destruction and immune reactions to insect products. Saliva of these insects provides a complex pharmacological armamentarium to block these vertebrate reactions. With the advent of transcriptomics, the sialomes (from the Greek word sialo = saliva) of at least two species of each of these families have been studied (except for the frog feeders), allowing an insight into the diverse pathways leading to today’s salivary composition within the Culicomorpha, having the sand flies as an outgroup. This review catalogs 1288 salivary proteins in 10 generic classes comprising over 150 different protein families, most of which we have no functional knowledge. These proteins and many sequence comparisons are displayed in a hyperlinked spreadsheet that hopefully will stimulate and facilitate the task of functional characterization of these proteins, and their possible use as novel pharmacological agents and epidemiological markers of insect vector exposure.

Graphical abstract

Research highlights

► Over 1200 salivary proteins of mosquitoes, black flies, biting midges and sand flies are analyzed. ► These proteins were grouped into over 150 different protein families, most of which we have no knowledge of their function. ► This work provides a platform for discovery of novel pharmacologically active agents and antimicrobial peptides. ► The fast evolution of these proteins is discussed within the context of the evolution of blood feeding within the Nematocera suborder.

Introduction

The blood-feeding habit evolved independently several times among different insect orders, and even within insect orders, such as the Diptera (Ribeiro, 1995). Among the several challenges associated with this peculiar diet, the vertebrate response against blood loss, the hemostasis process, represents a formidable barrier to efficient blood feeding. Within seconds of vessel laceration, mammalian platelets adhere to each other, forming a plug, and produce or expose pro-clotting and vasoconstrictory substances. Immune reactions can lead to mast cell degranulation and the release of biogenic amines (mainly histamine and serotonin) and eicosanoids (mainly leukotriene C4 and prostaglandin D2) that induce host itching reactions and edema that can prevent feeding or even kill the micropredator. Perhaps for these reasons, insects evolved a salivary concoction that disarms their host’s hemostasis and inflammation. Because vertebrate hemostasis and inflammation are complex and redundant, hematophagous insect saliva is also complex and redundant, containing dozens of active compounds (Ribeiro and Arca, 2009). Because this feeding mode evolved independently in several insect orders and families, the salivary composition among insects is typical of a convergent evolutionary scenario (Mans and Francischetti, 2010). On the other hand, the diversity among different genera within a same family has been also found to be large possibly due to the vertebrate host immune pressure over the salivary products, or due to the appearance of new hemostatic challenges that are posed by the evolving host genomes, such as the appearance of platelets in mammals, much more efficient than, for example, bird thrombocytes (Didisheim et al., 1959).

The Diptera were classically divided into two suborders, Nematocera and Brachycera, both of which have hematophagous flies, such as the mosquitoes, sand flies and black flies in the Nematocera, and horse flies, tsetse and stable flies, for example, in the Brachycera. This traditional division in two suborders is challenged because the Nematocera is considered paraphyletic regarding Brachycera (Yeates and Wiegmann, 1999), being replaced by several infra-orders, including the the Psycodomorpha that includes the family Psychodidae (sand flies), and the Culicomorpha on the other, thus postulating two independent events of blood feeding in the non-Brachycera flies (Null hypothesis). Notice that the common usage of Nematocera in this paper should be understood as “non-Brachycera” according to the current phylogenetic view. The Culicomorpha clade may have originated from a single blood-feeding ancestor during the Triassic, over 200 million years ago (MYA). This ancestor gave rise to the 11 extant families, 4 of which retain blood feeding, namely the Culicidae (mosquitoes), Simuliidae (black flies), Ceratopogonidae (biting midges) and Corethrellidae (frog biting midges) (Grimaldi and Engel, 2005). One family, the Blephariceridae (the net-winged midges), actually feeds on insect hemolymph, not vertebrate blood, and the remaining families (Chaoboridae, Dixidae, Niphomyiidae, Chironomidae, Thaumaleidae and Deuterophlebiidae) lost the blood-feeding habit (Grimaldi and Engel, 2005). Within the Nematocera hematophagy is restricted solely to the adult stage, and in most cases solely to the adult female stage, such as in mosquitoes and sand flies, where it is essential for egg development. These insects, in addition to blood, will also take sugar meals, which energize flight and basal metabolic needs; accordingly, their saliva will reflect adaptations to sugar feeding as well, including the presence of antimicrobial compounds.

On the other hand, given the extensive losses proposed in blood-feeding lifestyles of the Culicomorpha an even more parsimonious scenario would be that hematophagy evolved in the last common ancestor of the Psychodomorpha and the Culicomorpha, with extensive losses in hematophagous behavior across a number of lineages. Alternatively, blood-feeding behavior could have evolved independently in each major family of the Nematocera (Mans and Francischetti, 2010, Pawlowski et al., 1996, Ribeiro, 1995).

Methodologies developed in the past 10 years created an unprecedented window from which the transcription repertoire of particular organs can be brought to light. In the case of blood-feeding Nematocera, representative sialotranscriptomes (from the Greek sialo = saliva) exist from 4 of the 5 blood-feeding families, excluding the Corethrellidae (Table 1). With the exception of the frog biting flies, there are two or more sialotranscriptomes available for analysis for all other families of blood sucking Nematocera (Psychodidae, Culicidae, Ceratopogonidae and Simuliidae) allowing for an insight into the evolution of blood feeding within the Nematocera.

Using the information from the sialotranscriptomes indicated in Table 1, which includes the non-blood sucking mosquito Toxorhynchites amboinensis, a total of 1280 proteins were retrieved from GenBank and from our previous annotation. These proteins were grouped either (i) by comparing their primary sequence against each other by the tool Blast and producing groups of sequences with a particular threshold of identity over 40% of their length (note that this relatively small value was chosen because many sequences are fragments or truncated) (columns CL to DH of Table S1); or (ii) grouped following Psiblast at different levels of the switches –h and –j determining the e value threshold for inclusion of matches and number of iterations, respectively (columns DI to DN of Table S1) (Altschul et al., 1997). The sequences obtained were automatically aligned with the program ClustalX (Thompson et al., 1997) if less than 100 sequences were found in a particular group. Sequences were also submitted to several servers of the Danish Center for Biological Sequence Analysis to obtain indications of secretory signals (signalP, secretomeP and targetP servers) (Bendtsen et al., 2004, Emanuelsson et al., 2000, Nielsen et al., 1997, Sonnhammer et al., 1998), transmembrane domains (tmhmm server) (Sonnhammer et al., 1998) and mucin-type galactosylation (NetOglyc server) (Julenius et al., 2005). The data was transferred to an Excel spreadsheet and manually annotated to help classification of the various protein families (Supplemental Table S1). Notice that Table S1 may be somewhat redundant by including proteins that are >95% similar among themselves, but on the other hand may provide indication of alleles. These can be easily picked up on columns DE or DG of Table S1.

As mentioned above, the salivary composition of blood-feeding insects is quite complex and varied, containing various enzymes, proteins with various protease inhibitor domains, mucins, kratagonists (chelators of agonists) (Ribeiro and Arca, 2009) and a large number of proteins that are only found in blood sucking insects, as indicated by comparisons of their primary structure to the non-redundant protein database (NR) of the National Center for Biotechnology Information (NCBI). Most proteins of this last group have unknown function, as will be indicated below.

Annotation of Table S1 grouped the 1280 proteins in 10 main classes representing 155 different protein families (excluding the 106 orphan proteins), summarized in Table 2. These classes are organized in a gradient regarding a possible functional perspective, starting with enzymes (170 proteins in 23 families) and proteins with typical protease inhibitor domains, such as serpins or Kunitz-domain containing polypeptides to proteins with unknown function, many of which are uniquely found within this 1280 set.

A breakdown of the classes shown in Table 2 indicates the inner complexity of each class, and how much is known about the function of each subclass (Table 3). Most of the protein families have no known function; only 20 of the 155 families have been studied so far with at least one published work available. Below follows a description of the different families, which should serve as a guide for browsing Supplemental file S1.

Section snippets

Enzymes

Apyrase: Blood sucking Nematocera were known to hydrolyze ATP and ADP (Cupp et al., 1994, Cupp et al., 1995, Marinotti et al., 1996, Perez de Leon and Tabachnick, 1996, Reno and Novak, 2005, Ribeiro et al., 1989, Ribeiro et al., 1985, Ribeiro et al., 1986, Ribeiro et al., 1984), which are important hemostasis and inflammatory agonists released by platelets and broken cells following tissue injury. ADP is a potent inducer of platelet aggregation, and ATP induces neutrophil aggregation and

Antigen 5 family

This is a ubiquitous protein family belonging to the wider CAP superfamily (Gibbs et al., 2008). Its members are associated with host defenses in plants, and various functions in animals, such as toxins in snake and lizard venoms (Nobile et al., 1996, Yamazaki et al., 2002), proteolytic activity in Conus snails (Milne et al., 2003), and platelet aggregation inhibition in a tabanid salivary protein (Xu et al., 2008), although this latter function results from the novel incorporation of an RGD

Ubiquitous insect protein families existing outside Nematocera, function unknown

This group of proteins possibly contains housekeeping proteins that could be performing a function in the ER or Golgi, or may have been recruited to perform a salivary function.

30 kDa antigen/Aegyptin

Members of this protein family are found in the sialotranscriptomes of mosquitoes and black flies where they are abundantly expressed, and also in sand flies albeit at lower expression levels. They were first identified as a 30 kDa antigen in Aedes mosquitoes (Docena et al., 1999, Simons and Peng, 2001). Proteins of this family from An. stephensi and Ae. aegypti were shown more recently to inhibit platelet aggregation by interfering with collagen recognition (Calvo et al., 2007b, Yoshida

Found in both culicines and anophelines

The 56 kDa protein family is expressed in adult mosquito salivary glands, including male An. gambiae mosquitoes (Calvo et al., 2006b). RT-PCR experiments in Ae. aegypti and Ae. albopictus also revealed this transcript in female salivary glands and in males, indicating a function associated with sugar feeding or as an antimicrobial. Four iterations of psiblast (Altschul et al., 1997) retrieves bacterial proteins in addition of mosquito proteins (Arca et al., 2007, Arca et al., 2005, Ribeiro

Proteins so far found only in black flies

Two sialotranscriptomes were done so far for black flies, one with the North American autogenous species S. vittatum (Andersen et al., 2009) and the other with the South American S. nigrimanum (Ribeiro et al., 2010). From these 2 transcriptomes, 24 protein families were identified as exclusively found in black flies. Only one of these proteins has a known characterized function, the salivary vasodilator named SVEP for S. vittatum erythema protein (Cupp et al., 1998). These proteins families

Proteins so far found only in sand flies

Eight protein families were found unique to sand flies. Two of the families have been functionally characterized, including the vasodilatory peptide Maxadilan, specific of the genus Lutzomyia and the 33 kDa family, found in both New World and Old World genera and identified as a FXa clotting inhibitor (J. Valenzuela, unpublished). The remaining families have no known function. Interestingly, the 27–30 kDa family of P. argentipes, consisting of 4 related proteins (Anderson et al., 2006),

Proteins so far found only in biting midges

Eight protein families are exclusive to biting midges, including a quite expanded family of proteins in the range of 14–15 kDa, found in both C. sonorensis and C. nubeculosus. No member of any of the families has been characterized functionally.

Orphan proteins of conserved families of unknown function

Spreadsheet S1 provides links to 43 protein sequences found in sialotranscriptomes that have no match to other proteins described in sialotranscriptomes, but match proteins deposited in the NR database. These proteins may have been uniquely co-opted for a salivary function in the named insects, or may be poorly expressed housekeeping transcripts that were revealed by chance in the transcriptomes.

Orphan proteins of unique standing

An additional 63 proteins from various insects and containing a signal peptide indicative of secretion are described in Spreadsheet S1 which have neither matches to any sialotranscriptome, nor matches to known proteins in the NR database.

Evolutionary considerations

The evolution of the blood sucking habit in the Nematocera may have occurred at least twice, over two hundred million years ago, producing today’s sand flies and all the families within the Culicomorpha (the null evolutionary hypothesis) (Fig. 4). The salivary glands of these insects incorporate the evolutionary process toward this adaptation, a trajectory that included a menu initially composed mainly by dinosaurs and reptiles, changing to mostly birds and mammals after the extinction of the

Concluding remarks

Due possibly to host immune pressure, the evolutionary speed of salivary gland genes is at a fast pace. As such, it is remarkable that some common protein families involved in blood feeding and secreted in large amounts still exist in organisms that diverged over 150 MYA, as represented by the D7 protein family of mosquitoes, or the 5′-nucleotidase family of apyrases in the Culicomorpha. To the extent that these genes cannot mutate fast enough or keep a state of balanced polymorphism, they

Acknowledgements

This work was supported in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA. Because J.M.C.R. is a government employee and this is a government work, the work is in the public domain in the United States. Notwithstanding any other agreements, the NIH reserves the right to provide the work to PubMedCentral for display and use by the public, and PubMedCentral may tag or modify the work consistent with its

References (174)

  • R. Charlab et al.

    The invertebrate growth factor/CECR1 subfamily of adenosine deaminase proteins

    Gene

    (2001)
  • O. Emanuelsson et al.

    Predicting subcellular localization of proteins based on their N-terminal amino acid sequence

    J. Mol. Biol.

    (2000)
  • E. Faudry et al.

    Triatoma infestans apyrases belong to the 5′-nucleotidase family

    J. Biol. Chem.

    (2004)
  • D. Ferrandon et al.

    Sensing infection in Drosophila: toll and beyond

    Semin. Immunol.

    (2004)
  • A. Fradkov et al.

    Enzyme from the medicinal leech (Hirudo medicinalis) that specifically splits endo-epsilon(-gamma-Glu)-Lys isopeptide bonds: cDNA cloning and protein primary structure

    FEBS Lett.

    (1996)
  • I.M. Francischetti et al.

    Cloning of a salivary gland metalloprotease and characterization of gelatinase and fibrin(ogen)lytic activities in the saliva of the Lyme disease tick vector Ixodes scapularis

    Biochem. Biophys. Res. Commun.

    (2003)
  • A.L. Harvey

    Recent studies on dendrotoxins and potassium ion channels

    Gen. Pharmacol.

    (1997)
  • H. Isawa et al.

    Identification and characterization of a new kallikrein-kinin system inhibitor from the salivary glands of the malaria vector mosquito Anopheles stephensi

    Insect Biochem. Mol. Biol.

    (2007)
  • A.A. James et al.

    A salivary gland-specific, maltase-like gene of the vector mosquito, Aedes aegypti

    Gene

    (1989)
  • B. Jennings et al.

    A novel 25 kDa protein from the venom of Bitis arietans with similarity to C-type lectins causes fibrinogen-dependent platelet agglutination

    Toxicon

    (2005)
  • J.K. Johnson et al.

    Cloning and characterization of a dopachrome conversion enzyme from the yellow fever mosquito, Aedes aegypti

    Insect Biochem. Mol. Biol.

    (2001)
  • M.R. Kanost

    Serine proteinase inhibitors in arthropod immunity

    Dev. Comp. Immunol.

    (1999)
  • A.M. Korayem et al.

    A Drosophila salivary gland mucin is also expressed in immune tissues: evidence for a function in coagulation and the entrapment of bacteria

    Insect Biochem. Mol. Biol.

    (2004)
  • M. Kotsyfakis et al.

    Antiinflammatory and immunosuppressive activity of sialostatin L, a salivary cystatin from the tick Ixodes scapularis

    J. Biol. Chem.

    (2006)
  • M. Kotsyfakis et al.

    Selective cysteine protease inhibition contributes to blood-feeding success of the tick Ixodes scapularis

    J. Biol. Chem.

    (2007)
  • J. Krzywinski et al.

    Analysis of the complete mitochondrial DNA from Anopheles funestus: an improved dipteran mitochondrial genome annotation and a temporal dimension of mosquito evolution

    Mol. Phylogenet. Evol.

    (2006)
  • E.A. Lerner et al.

    Maxadilan: cloning and functional expression of the gene encoding this potent vasodilator peptide

    J. Biol. Chem.

    (1992)
  • E.A. Lerner et al.

    Isolation of maxadilan, a potent vasodilatory peptide from the salivary glands of the sand fly Lutzomyia longipalpis

    J. Biol. Chem.

    (1991)
  • F. Lombardo et al.

    Promoter sequences of the putative Anopheles gambiae apyrase confer salivary gland expression in Drosophila melanogaster

    J. Biol. Chem.

    (2000)
  • A. Lanfrancotti et al.

    Novel cDNAs encoding salivary proteins from the malaria vector Anopheles gambiae

    FEBS Lett.

    (2002)
  • F. Lombardo et al.

    The Anopheles gambiae salivary protein gSG6: an anopheline-specific protein with a blood-feeding role

    Insect Biochem. Mol. Biol.

    (2009)
  • B.J. Mans et al.

    The crystal structure of D7r4, a salivary biogenic amine-binding protein from the malaria mosquito Anopheles gambiae

    J. Biol. Chem.

    (2007)
  • B.J. Mans et al.

    Comparative sialomics between hard and soft ticks: implications for the evolution of blood-feeding behavior

    Insect Biochem. Mol. Biol.

    (2008)
  • O. Marinotti et al.

    An alpha-glucosidase in the salivary glands of the vector mosquito, Aedes aegypti

    Insect Biochem.

    (1990)
  • O. Marinotti et al.

    Diet and salivation in female Aedes aegypti mosquitoes

    J. Insect Physiol.

    (1990)
  • O. Marinotti et al.

    Apyrase and alpha-glucosidase in the salivary glands of Aedes albopictus

    Comp. Biochem. Physiol.

    (1996)
  • M. Abrahamson et al.

    Cystatins

    Biochem. Soc. Symp.

    (2003)
  • S. Albert et al.

    The family of major royal jelly proteins and its evolution

    J. Mol. Evol.

    (1999)
  • S.F. Altschul et al.

    Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

    Nucleic Acids Res.

    (1997)
  • M. Ameri et al.

    An immunoglobulin binding protein (antigen 5) of the stable fly (Diptera: Muscidae) salivary gland stimulates bovine immune responses

    J. Med. Entomol.

    (2008)
  • J.F. Andersen et al.

    Insight into the sialome of the black fly, Simulium vittatum

    J. Proteome Res.

    (2009)
  • J.M. Anderson et al.

    Comparative salivary gland transcriptomics of sandfly vectors of visceral leishmaniasis

    BMC Genomics

    (2006)
  • B. Arca et al.

    Trapping cDNAs encoding secreted proteins from the salivary glands of the malaria vector Anopheles gambiae

    Proc. Natl. Acad. Sci. U. S. A.

    (1999)
  • B. Arca et al.

    A cluster of four D7-related genes is expressed in the salivary glands of the African malaria vector Anopheles gambiae

    Insect Mol. Biol.

    (2002)
  • B. Arca et al.

    An updated catalogue of salivary gland transcripts in the adult female mosquito, Anopheles gambiae

    J. Exp. Biol.

    (2005)
  • P. Ascenzi et al.

    The bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor): a milestone protein

    Curr. Protein Pept. Sci.

    (2003)
  • D. Bahia et al.

    Antibodies from dogs with canine visceral leishmaniasis recognise two proteins from the saliva of Lutzomyia longipalpis

    Parasitol. Res.

    (2007)
  • A. Barral et al.

    Human immune response to sand fly salivary gland antigens: a useful epidemiological marker?

    Am. J. Trop. Med. Hyg.

    (2000)
  • I.P. Baskova et al.

    Destabilase, the novel epsilon-(gamma-Glu)-Lys isopeptidase with thrombolytic activity

    Blood Coagul. Fibrinolysis

    (1991)
  • B.T. Beerntsen et al.

    Characterization of the Sialokinin I gene encoding the salivary vasodilator of the yellow fever mosquito, Aedes aegypti

    Insect Mol. Biol.

    (1999)
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