Multispecies comparisons exploring the evolution of insect immunity

The availability of multiple sequenced insect genomes has greatly facilitated the dissection of their immune repertoires into functional modules and sequential phases of an integrated innate immune system response. Concomitant analysis of orthology, sequence variation, and functional data from experimentation has provided important insights towards understanding the principles that govern the ongoing evolution of innate immunity. The emergent picture is one of a robust architecture, shared to some extent even with vertebrates, but with diversified inputs and outputs. The observations that different immune modules have distinct and even contrasting evolutionary dynamics have helped explain the overall flexibility of a system capable of adapting to a multitude of new challenges.

The sequencing of the A. aegypti genome (Nene et al,, 2007) permitted a comprehensive comparison of the immune repertoires of two very different mosquito species, separated by approximately 150 million years of evolution (Waterhouse et al., 2007). A. aegypti is the major vector of dengue and yellow fever, as well as of several other important encephalitis-causing viruses, such as West Nile and Chikungunya. Thus, its successful adaptation to new, mostly urban environments presents a major challenge to global public health. In contrast, anopheline mosquitoes are infrequently associated with viral transmission, with O'nyong nyong being the only virus known to be transmitted primarily through A. gambiae and perhaps through Anopheles funestus. As A. aegypti can be infected with and transmit the avian parasite Plasmodium gallinaceum, as well as filarial nematodes that are also transmitted by Anopheles, comparative immunogenom-ics provides opportunities for studying a variety of mosquito-pathogen interactions, both common and unique.

Multi-species comparative immunogenomic studies of the two mosquitoes and the fruit fly that diverged from them approximately 250 million years ago, characterized and compared almost 1000 genes as components of immune-signalling pathways, or members of some 30 immune-related families or subfamilies (Waterhouse et al., 2007). Rigorous phylogenetic analysis across this set of immune-related gene families enabled the identification of single-copy orthologous trios, mosquito-specific pairs, and genes showing characteristics of family expansions and gene losses, or high sequence divergence that precluded confident determination of phylogenetic relationships. The prevalence of orthologous trios, mosquito-specific orthologous pairs, and species-specific genes varied widely among immune gene families, clearly indicating that families vary in their degree of diversification (Figure 6.3a). At one extreme, families of enzymes involved in oxidative defence, the A- and B-type scavenger receptors, and inhibitors of apoptosis show a high proportion of orthologues. The C-type lectins, which can act as opsonins or modulators of melanization, display an intermediate level of conservation: they show large expansions (particularly in Drosophila and Aedes) while retaining a set of at least nine orthologous trios. The modulators, serine protease inhibitors, show a high level of conservation between the two mosquitoes, whereas few have confident orthologues in Drosophila. At the other end of the scale, only a few families of AMPs are shared among the three species: the effectors

□ Species specific genes

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□ Species specific genes

□ Mosquito 1:1 orthologues

□ 1:1:1 orthologous trios

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Figure 6.3 (a) The repertoire of putative immune-related gene families identified in Drosophila melanogaster(Dm), Anopheles gambiae (Ag), and Aedes aegypti (Aa) show highly variable levels of orthology conservation. The total number of genes identified in D. melanogaster (first bar), A. gambiae (second bar), and A. aegypti (third bar) are summed from the numbers of 1:1:1 orthologous trios (black), mosquito-specific 1:1 orthologues (grey), and species-specific genes (white) for each gene (sub)family. Families are arranged from left to right according to the decreasing proportion of 1:1:1 orthologous trios within the family. See Table 6.1 for the full list of gene family acronyms and definitions. (b) Sequence divergence among fruit fly-mosquito orthologous trios. All identifiable single-copy trios were compared with the subset of immune-related single-copy trios in terms of genetic distances of each mosquito (A. gambiae or A. aegypti) protein to the corresponding D. melanogaster orthologue. The distance means for immunity and all trios (dashed and solid lines, respectively) indicate that immunity trios (black dots) are significantly more divergent than all trios (grey dots). Signal transducers (boxed) are among the most highly divergent trios, despite high levels of conservation in terms of orthology. DEF, defensin. Both panels are adapted from Waterhouse et al. (2007).

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Figure 6.3 (a) The repertoire of putative immune-related gene families identified in Drosophila melanogaster(Dm), Anopheles gambiae (Ag), and Aedes aegypti (Aa) show highly variable levels of orthology conservation. The total number of genes identified in D. melanogaster (first bar), A. gambiae (second bar), and A. aegypti (third bar) are summed from the numbers of 1:1:1 orthologous trios (black), mosquito-specific 1:1 orthologues (grey), and species-specific genes (white) for each gene (sub)family. Families are arranged from left to right according to the decreasing proportion of 1:1:1 orthologous trios within the family. See Table 6.1 for the full list of gene family acronyms and definitions. (b) Sequence divergence among fruit fly-mosquito orthologous trios. All identifiable single-copy trios were compared with the subset of immune-related single-copy trios in terms of genetic distances of each mosquito (A. gambiae or A. aegypti) protein to the corresponding D. melanogaster orthologue. The distance means for immunity and all trios (dashed and solid lines, respectively) indicate that immunity trios (black dots) are significantly more divergent than all trios (grey dots). Signal transducers (boxed) are among the most highly divergent trios, despite high levels of conservation in terms of orthology. DEF, defensin. Both panels are adapted from Waterhouse et al. (2007).

show hardly any maintenance of single-copy orthologues. High diversity is also observed within the lysozyme family of peptidoglycan-hydrolysing enzymes. This family has expanded independently in each species, leaving only one gene that encodes a multiple-lysozyme domain protein with a clear three-way orthologous relationship. Across the recognized immune-related gene families shown in Figure 6.3a, some have undergone species- or lineage-specific expansions or have suffered independent losses resulting in fewer identifiable cases of single-copy orthology; other families are more conservative and retain many clear orthologous relationships.

In addition to differences in orthology conservation, the immune repertoires show interesting patterns of sequence diversification. Previous observations of elevated divergence in immune genes were based on sequence identities between orthologous Drosophila-Anopheles pairs (Zdobnov et al., 2002). Multi-species analysis allowed more rigorous comparisons of immune and non-immune trios, in terms of the computed phylogenetic distances of each mosquito protein to their corresponding Drosophila orthologue. These distances revealed that immune trios display significantly higher levels of sequence divergence than the full set of identifiable trios in these genomes (Figure 6.3b). This three-way analysis detected several Anopheles immunity genes that appear considerably more divergent than their Aedes orthologues. This trend extends to the full set of trios and implies greater accumulation of amino acid substitutions in the conserved protein cores of Anopheles compared to Aedes. Remarkably, many components of signal transduction pathways show high conservation in terms of orthology (within Diptera and also other animal groups), but are in fact among the most highly divergent of the single-copy trios at the sequence level (Figure 6.3b). In contrast, multigene families with large species-specific expansions, for example C-type lectins (CTLs) and clip-domain serine proteases (CLIPs), retain orthologous trios that are highly conserved in sequence. Evidently, strict maintenance of copy numbers does not necessarily imply high conservation of protein sequence identities: these two measures represent distinct evolutionary processes.

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