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) (). 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 dinosaurs and irradiation of mammal 65 MYA. Mammals “invented” the blood platelet, creating a major barrier to blood feeding, or at least creating the opportunity for insects to develop new pharmacological agents to disarm platelet function that increase their fitness by increasing blood uptake success. Comparative sialotranscriptome analysis allows a snapshot of this evolutionary process over a long evolutionary time, and allows some insight into the Culicomorpha “Eve”, with sand flies as an outgroup.
Fig. 4 Blood-feeding evolution in the Nematocera. Indicated in red are the Null hypothesis (independent evolution of blood-feeding behavior in the Psychomorpha and Culicomorpha) presented in this study, according to Grimaldi and Engel (2005). Numbers at the (more ...)
But before we launch into this exercise, here are some difficulties: First, when attempting to visualize the evolutionary scenario to hematophagy starting with the Culicomorpha or Psycomorpha “Eve”, we contemplate that the majority of the proteins discovered so far have no known function, and many have no similarities to any other known proteins. It is fair to say at this point in time that the overwhelming majority of salivary proteins in the Nematocera have no known function, as indicated in . Even if the protein family is well known, we may not “a priori” determine its function; for example, Kazal-domain containing proteins may function as a serine protease inhibitor, and thus could have anti-clotting function, but can also be antimicrobial. For this reason, in species where only females blood-feed, for example in mosquitoes, comparative transcriptomes analysis between male and females can indicate those that are adaptive to sugar feeding, if they are found in male salivary glands. It is thus important to determine whether a protein functions within a blood feeding or a sugar feeding context to ascribe evolutionary pathways associated with hematophagy. Secondly, large expanded families may have multiple functions. For example, a few years ago we were completely ignorant on the function of the D7 family in mosquitoes. We learned that they mainly function as kratagonists, in some cases antagonizing serotonin, in other cases histamine and in other cases leukotrienes or thromboxane A2
(Calvo et al., 2009a
; Mans et al., 2007
); but it can also function as an inhibitor of bradykinin formation (Isawa et al., 2002
). Identification of orthologs helps in assigning functions, and from their alignment, models can be built for functional identification in new organisms, but we cannot a priori determine the specificity of the more distant members, where novel functions may be acquired. Thirdly, it appears that the salivary gland genes are at a relatively rapid pace of evolution, leading to the appearance of protein families that are unique even at the genus level of some families, or even at the subgenus level, as is the gSG7 protein of anophelines. This fast evolution is probably a response to the immune pressure posed by their hosts. Perhaps for this last consideration, very few salivary protein families are conserved in all Culicomorpha, allowing for only a few unique genes common to all members that resisted the passage of time. And these few genes could have been the product of convergent evolution and not common ancestry.
A most probable common mode of feeding of the adult Nematocera “Eve,” from which both sand flies and Culicomorpha were derived, must have been a plant feeding behavior, surely not on flower nectar, because these did not exist at 200 MYA. This may explain the ubiquitous presence of multiple sugar hydrolyzing enzymes such as amylase and maltase, common to sand flies and Culicomorpha. The same can be stated for antimicrobial peptides such as the ubiquitous lysozyme and other common antimicrobial peptide domains, as they might have controlled microbial growth in the insect's crop. This set of salivary proteins should be common to all Nematocera, blood feeding or not.
Genes coding for ubiquitous proteins, such as antigen 5 or trypsins, are difficult to assign an evolutionary scenario without a clear characterization of their orthologous function in different families. For example, if salivary antigen 5 proteins across the different families are shown to retain a immunoglobulin binding function, or if trypsins are found to have a conserved fibrinolytic function, these orthologous functions could make a strong point on their association with the evolution of blood feeding. But these genes might otherwise be associated with innate immunity and not related to hematophagy. Their functional characterization is important in this evolutionary context, in addition to their possible function in immunity and blood feeding.
From an evolutionary (not biochemical) viewpoint, the assignment of proteins to a specific protein family obviously holds the implication that all members of such a family are homologous. That is, that all members derived from a common ancestral fold that originated once in the distant past. In the case where such families occur universally throughout the animal kingdom, their presence in sialomes per se
cannot be taken as evidence of common blood-feeding origins. These protein families reside in most genomes and could have been recruited to the sialome at any given time in the evolution of blood-feeding behavior. This would certainly be the case for the majority of Class I families such as the enzymes, protease inhibitor domains and anti-microbials. Results do indicate, however, that different arthropod lineages and families possess unique sialome compositions (Mans and Francischetti, 2010
). This corresponds to lineage specific innovations directly related to adaptation to a blood-feeding lifestyle and do indicate that specific families responded in unique ways to the challenge of the blood-feeding interface. What is of interest though, is that the various lineages share very few of the protein families common outside the Nematocera and no indication can be found that there is a lineage specific association of protein families as might have been expected if the various lineages share common blood-feeding histories (). The fact that common protein families are found in closely related lineages is certainly indicative of a shared history, but the ways these protein folds have been exploited to evolve novel functions are what is remarkable and significant (Mans and Francischetti, 2010
In the elucidation of molecular origins, the accurate identification of orthologous proteins, rather than protein folds is crucial. Orthologous proteins are related by vertical descent and generally possess similar functions and mechanisms that are conserved over time due to negative selection. While immune pressures and gene losses can certainly account for some of the variations observed in the association of protein folds and function, an analysis of the data clearly show that orthologous proteins (with conserved function) are found within the various nematocerous families and that within these lineages immune pressure may play a relatively small role in determining functional
switches (but could lead to gene function loss and gene elimination). In many cases, these functions have been conserved for more than 100–200 million years. An example is the biogenic amine binding D7-fold found in mosquitoes with conserved binding mechanisms, but known to be highly immunogenic (Calvo et al., 2009a
; Mans et al., 2007
The review thus far considered the sialomes from the perspective of the distribution of protein families in and outside the Nematocera and their possible functions. As such, there are a number of protein families shared between all Nematocera that is also found in other arthropods and a number of 74 families that are specifically nematocerous (, ). Only two of these 74 unique families are shared between the Culicomorpha and sand flies, the 41 kDa superfamily, possibly associated with sugar feeding, and the 30 kDa/Aegyptin family, associated with blood feeding (). As we move into the Nematocera lineages, the number of shared families increase, but even so it is only near 50% of the totality of unique families () indicating many recent acquisitions of novel families, or evolution of old families to such a degree that they are not recognizable by sequence similarity analysis. As such, the argument could have been made that blood-feeding evolved in the last common ancestor of the Culicomorpha and Psycomorpha, with subsequent lineage specific innovations after speciation of the main families. However, most of the protein families that are shared between the nematocerous families are ubiquitous and found widely in other blood-feeding and toxic arthropods as well (Fry et al., 2009
; Mans et al., 2008
; Mans and Francischetti, 2010
). Those that have been exapted from house-keeping functions to function at the host-vector feeding interface could have been recruited at any stage and their use as indicators of common blood-feeding origins seems tenuous. A nice example of such proteins are the 5'-nucleotidases, which have been recruited several times in different unrelated blood-feeding arthropods that include kissing bugs, mosquitoes and ticks (Champagne et al., 1995
; Faudry et al., 2004
; Stutzer et al., 2009
); accordingly, the common finding of 5'-nucleotidases serving an apyrase function in Culicomorpha could have been acquired by independent recruitment of this gene family by the different lineages. In the same line, the recruitment of the OBP superfamily by both Culicomorpha and sand flies could have been the result of convergent evolution. Elucidation of the function and crystal structure of these sand fly proteins may throw more light into this issue.
The data obtained, even if extensive gene losses and continuous recruitment of new folds are considered, does not give overwhelming support for the Null hypothesis. In the light of the above exposition, the possibility that the various blood-feeding Nematoceran families evolved blood-feeding behavior independently should be considered seriously, even if this seems to be the least likely parsimonious scenario (Mans and Francischetti, 2010
). Of interest, would be the fact that this is the third group of closely related organisms (the other two being ticks and triatomine bugs) that posses similar folds, but on closer analysis would seem to indicate independent evolution of functions involved at the blood-feeding interface. Previous observations with triatomine bugs from the genera Rhodnius and Triatoma, as well as soft and hard tick families, suggest that these organisms evolved hematophagous behavior independently (Mans et al. 2008
; Mans and Francischetti, 2010
). The question is raised why closely related organisms are prone to evolve blood-feeding behavior independently. In this regard, previous similar lifestyles that would predispose towards blood-feeding (plant feeding, predation of insects, scavenging of arthropod hemolymph), in which birds and mammals (and possibly dinosaurs) suddenly occupies the same environmental niche in which non-hematophagous arthropods were living, could have precipitated an association of vertebrates and the previous food niche, thus causing an initial exploration of vertebrates as a potential food source.
Within this discussion, it is puzzling that there is no common salivary anticlotting protein in the Culicomorpha. Blood clotting is relatively conserved in vertebrates, all having thrombin as a final enzyme that processes fibrinogen to fibrin. This clotting system is quite ancient and must have posed a barrier to blood feeding by the ancestral hematophagous Culicomorpha. Nonetheless, Culicoides and black flies abound with Kunitz-domain proteins (yet to be demonstrated to be anti-clotting), a black fly anti-thrombin is member of the OBP family, Culicine mosquitoes have a serpin that inhibits factor Xa, and anopheline mosquitoes have a unique anti thrombin named anophelin (see references in ). No conservation of an anti-clotting is verified even at the Culicidae level. It has been observed (Ribeiro, unpublished
) that salivary gland homogenates (SGH) of Ae. aegypti
failed to increase “in vitro” recalcification clotting time of plasma derived from mosquito exposed Guinea pigs, and also the plasma of one of the authors (JMCR), who used to feed mosquitoes on himself at the time; however, elevated recalcification time was observed with SGH-treated plasma derived from unexposed mammals. It appears that the value of the Aedes serpin is clearly under pressure to either be less immunogenic or for Aedes to “find out” something else better suited to the job. Better documented is the case of the salivary vasodilatory peptide from L. longipalpis
named maxadilan, shown to be immunogenic; host immunity was associated with less fly blood ingestion and egg output (Milleron et al., 2004a
; Milleron et al., 2004b
). Perhaps for this reason, maxadilan genes from wild populations are very variable at the amino acid level, with up to 23% amino acid divergence between alleles (Lanzaro et al., 1999
). Interestingly these alleles have different cross reactivity to antibodies, but equal vasodilatory activity (Milleron et al., 2004b
) indicating the adaptive value of this polymorphism which reflects antigenic variation, not functional improvement. A detailed population genetics work on salivary genes involved in blood feeding will certainly enhance our vision on the evolutionary paths leading toward this peculiar diet. The lack of a common salivary anti-clotting agent among the Culicomorpha could thus support either the renunciation of the null hypothesis, or represent fast gene turnover within a commonly derived hematophagous lineage.