IB404 - 12 - Other arthropods – Feb 271. Mosquitoes of the genus Anopheles are the major vectors of malaria around the world, with about 300m cases per year, while A. gambiae is the primary vector in Africa, where about 1m deaths, mostly children, occur each year, plus lots of morbidity.

2. Plasmodium resistance to quinone and Anopheles resistance of insecticides, and opposition to DDT-spraying, has led to a resurgence of malaria in Africa, with a particularly virulent and lethal form of Plasmodium causing considerable concern.

3. Two Plasmodium genomes and the Anopheles gambiae genome were completed and published in 2002. We may talk about Plasmodium later. Anopheles was sequenced by Celera using a 12X WGS strategy, funded by the NIH-National Institutes of Allergy

and Infectious Diseases.

4. The genome is about 250 Mbp with about 14,000 genes. Obviously the major comparisons to be made were with Drosophila melanogaster, and these two major lineages of flies (representing the two major suborders of Diptera) are thought to have diverged roughly 250 Myr ago (in the Permian, before the mass extinction that ended the trilobites, and mammals split from reptiles).

5. The first observation is that roughly half the genes, about 6000, are still 1:1 orthologs, simply diverged in sequence since then, with the average amino acid identity down to around 55%, although ranging from about 20-100%. Another ~1000 genes encode proteins that have complicated 2:1, 3:1, 4:1 andhigher relationships, that is, there areparalogs in one or both species resultingfrom gene duplications in either lineage.

6. Another ~2000 genes encode proteins with weak matches in the other fly and other insects, while another ~1000 have matches in non-insect species - these may be lost from one fly.

7. Finally 2-3000 genes encode proteins with no matches in other genomes so are species-specific. These must be rapidly evolving proteins with ecologically or sexually relevant roles to be evolving so fast. They are also shorter, and may not be well annotated.

8. Within the ~6000 1:1 orthologs it is possible to discern trends in terms of which classes of proteins are most conserved versus rapidly evolving. The most conserved are structural proteins, like actins and tubulins, while the most rapidly evolving even though not duplicated, are proteins involved in immunity and defense. And, of course, the 50% UNKNOWN category.

9. Anopheles has three chromosomes, and remarkably, when they mapped where all these 1:1 fly orthologs were on the chromosome arms, it turns out that despite a lot of gene movement between arms, the basic identity of the five chromosome arms can still be recognized (unfortunately, except for the X, they have different names). That is, the arms have stayed intact through 250 Myr of evolution in each lineage so there is still a lot of synteny (shown by colors in diagram). Presumably there have been innumerable paracentric inversions within the arms, but very few pericentric inversions that would mix the arms, and relatively few translocations or transpositions between different chromosomes. The autosomal arms themselves have not even been re-associated with each other, e.g. 2L and 2R in Drosophila are now 3R and 3L in Anopheles, although this may just be chance, because such re-assortments are known even within the genus Drosophila.

10. There is also still considerable microsynteny, that is, genes in the same orientation and order on segments of chromosomes, but it never extends very far before some kind of chromosomal rearrangement has reorganized segments of genes. A few large clusters remain, like the homeotic genes in the HOX cluster.

11. Several gene families were identified as considerably larger in the mosquito, and these commonly involved local expansions or clusters of genes, for example, this family of FBN domain proteins, thought to mediate binding to bacteria, perhaps in the gut in association with the bloodmeal. Notice that there are just two instances of 1:1 orthologs in this gene family (near the top of the tree), the rest are species-specific expansions, primarily in Anopheles. And most of these expanded subfamilies of genes are in tandem arrays in particular sites in the genome (indicated by the lines to the chromosomes on the right).

Other gut enzymes are also somewhat expanded, e.g. serine proteases and exo- and endo-nucleases, again presumably all to do with digesting the large bloodmeal.

12. Many other rapidly evolving gene families show similar, if less uneven, species-specific gene subfamily expansion. For example, cytochrome p450s involved in detoxification of all sorts of xenobiotics, including insecticides, and immune system genes involved in destroying bacteria. I worked up all the chemoreceptors involved in olfaction and taste and found the same kinds of things. The example here is the odorant receptors. Most of the Anopheles receptors in red form discrete clusters in this tree, as do most of the black Drosophila receptors. Only two 1:1 orthologs remain, for example at the very bottom of this tree. This protein happens to be a obligate heterodimer partner for all of the others, so it can’t evolve very quickly (notice short branches).

Anopheles subfamily expansion

Drosophila subfamily expansion

Conserved orthologs

13. Many transcriptome and proteome studies have now been done with this genome. Here is a simple one comparing un-fed versus blood-fed females (after 1-3 days when they are converting the blood meal into eggs). The classes of up- and down-regulated genes match the biology, for example, muscle and other structural genes are down-regulated because the mosquito is not actively flying and seeking hosts, while digestive enzymes, lipid transporters, and synthetic pathways were up-regulated, presumably facilitating conversion of the blood meal into eggs.

Aedes aegypti is the major vector of dengue fever today, but is commonly called the yellow fever mosquito because it vectored that before it was largely eliminated by vaccination (which works poorly for dengue because of the many viral variants). Its genome turned out to be huge, around 1.4 Gbp or half the size of ours, and was a real struggle for WGS sequencing. The assembly therefore has many more gaps making it harder to work with.

Aedes aegypti

The larger size is mostly the result of accumulation of a huge diversity of transposons, shown on right in pie chart. Note that only one third of this genome is single copy DNA, which is the genic portion. MITES are small versions of DNA transposons, while SINES are small versions of retrotransposons.

The Culex pipiens genome (vector of West Nile virus) is done. 12 more Anopheles are starting.

Bombyx mori, the domesticated silk moth from Asia, was the first lepidopteran genome sequence available (Japanese and Chinese papers in 2004, and joint in 2009). It is around 500 Mbp, 50% transposons, with again around 15,000 genes annotated. Without belaboring all the details, you can imagine their identification of many genes involved in producing silk, as well as the usual arrays of odorant receptors, cuticle proteins, defense genes, etc.

Bombyx mori

The Beijing Genome Institute, now known simply as the BGI, then re-sequenced 40 wild and domesticated strains using ILLUMINA, each to 3X coverage, to identify SNPs (single nucleotide polymorphisms) and indels (insertions and deletions) amongst them by lining these up with the reference sequence (the closest form of comparative genomics, becoming population genomics, something now done with humans too). They find remarkably that the wild strains (green) are only slightly more polymorphic than the domesticated strains from all over the world. Somehow domestication was achieved without a bottleneck?

Tribolium castaneum is called the red flour beetle because it is a pest of stored grains. It is also the second best developed molecular genetic model system in insects after Drosophila melanogaster, with good RNAi, mutants, transformation, and ease of maintenance of strains. As a result it has become a major system for comparative evo-devo studies, especially of the early developmental and HOX complex genes. Its genome is also nice and small at just 160 Mbp, only 30% transposons, and ~16,000 nice small compact genes and small introns (unlike Aedes and Bombyx genes with long introns).

Tribolium castaneum

I’m jumping ahead here, but including the honey bee Apis mellifera in the analysis, the authors of this paper show that while the vast majority of the clear ~6700 orthologous genes that these insects share with us humans are shared by all three insect genomes (center of Venn diagram), each group has some genes uniquely shared with us, usually around 100-200. Thus some of these genes cannot be studied in Drosophila as they have been lost from flies. We’ve studied several, e.g. telomerase and a novel opsin lineage, plus the entire DNA methylation system.

The honey bee genome is ~250 Mbp and we conservatively annotated ~12,000 genes. We are currently upgrading the genome assembly with 454 and ILLUMINA sequence, plus using deeper 454 sequencing of cDNAs and comparisons with dwarf honey bees and bumble bees to identify additional genes. It has many remarkably divergent features, some of which make sense in light of its radically divergent ecology.

Apis mellifera

1. Its genome is a mosaic of AT- and GC-rich regions, with most short conserved genes in the AT-rich regions. We have no idea what this is about, essentially the reverse of our genome.2. It has essentially no retrotransposons, and only 1% of the genome is DNA transposons. Again we haven’t a clue why. It is not a feature of Hymenoptera or haplo-diploidy because Nasonia vitripennis, a parasitoid wasp, has lots of retrotransposons, as do the ants we are doing now.3. 15 of its 16 chromosomes are acrocentric (like the Drosophila X), with the centromere at one end, while most insects have mostly metacentric chromosomes - no idea why.4. It has the highest recombination rate of any known organism, ~10X ours, and don’t know why.5. Its repertoire of odorant receptors has expanded, to around 170 compared with 60-100 in flies and moths, presumably mediating perception of floral odors and several pheromone blends.6. Its repertoire of gustatory receptors and detoxification enzymes like p450s is much smaller compared with flies and moths and beetles and wasps, apparently because of its mutualistic relationship with plants (doesn’t need to handle nasty defensive plant secondary compounds) and inactive larval stage (fed by the workers). Both loss of gene lineages (presumably by deletion) and failure to amplify others (presumably by failing to retain duplicated genes) have occurred.

Gene Robinson won a $2.5m NIH Pioneer Award in 2009 and is now sequencing 10 more bees.

Acyrthosiphon pisum and Pediculus humanusThese are the first two non-metamorphosing insects to be sequenced. Again they reveal all sorts of interesting genome biology, especially with regards to their obligate bacterial endosymbionts that facilitate their remarkable “parasitic” lifestyles, and which were sequenced along with them.

The aphid genome is ~500 Mbp and they annotated ~30,000 genes, but 10,000 of these are only ab initio predictions and 10,000 are aphid-specific. The most interesting result, perhaps, is that the aphid genome has lost most of the genes involved in enzymatic pathways producing and inter-converting amino acids, while its obligate endosymbiont Buchnera aphidicola, has all of these pathways. Bizarrely enough, the aphid has a few of these enzymes that the bacterium has lost, making this symbiotic relationship mutually obligate and allowing aphids to live on plant phloem or sap that is low in amino acids.

The louse genome is the smallest so far at 110 Mbp, and concordant with this it has lost a lot of genes, down to ~11,000, presumably resulting from its obligate parasitic lifestyle. For example, it has only 10 odorant receptors and only 10 gustatory receptors, as well as far fewer detoxification enzymes that any other insect. It too has an obligate endosymbiont bacterium, called Riesia pediculicola, which like Buchnera has a highly reduced genome of ~600 genes, mostly in a single linear chromosome. Remarkably it also has a large circular plasmid which encodes the enzymatic pathway for vitamin B5 (pantothenic acid), which is deficient in the louse diet of human blood.

This tree of the arthropods targeted for genome sequencing from the bee genome paper in 2006 is a little out of date (red was published, blue was draft, and green was in progress), but it shows the major lineages. The Daphnia pulex (crustacean water flea) genome is pretty amazing, encoding at least 30,000 and maybe 40,000 genes, apparently because of the complex aquatic environments it inhabits (several “species” in one genome!). The Ixodes scapularis deer tick (vector of Lyme disease) genome is huge at around 2.2 Gbp, and the Rhodnius prolixus kissing bug (vector of Chagas disease) is also unwieldly large (1 Gbp), hence they are both lagging.

The small migratory ground bird known as the American woodcock returns to this area early each spring and the males perform spectacular display flights at dusk on prairie patches around town. The easiest place to watch them is Meadowbrook park; dress warmly and go to the west parking lot on South Race Street, about 100 yards beyond Windsor Rd on the left, just after the Lindsley Clark retirement home. Displays start around 6 and by 6:30 it is too dark to see anything. It's best to get there early and walk along the path to the east to a bench next to an old wagon looking south. Sitting there allows you to see the birds against the still somewhat light sky. You will hear the males making a loud "neep" call on the ground (reminiscent of the courtship call of the nighthawks that will soon return as well), and then take off on a high circling twittering flight before spiraling down again to repeat the performance. Females can sometimes be seen flying by taking in the shows, and will mate with their chosen male(s) before nesting alone.

Woodcock courtship displays