Gut Symbionts in Arthropods

Insects may contain complex and diverse societies of microbes in their guts, yet relatively little is known about how these resident microbes shape the physiology of their hosts (Cazemier et al. 1997, Kaufman et al. 2000). The primary habitat for microorganisms associated with insects is the hindgut. The termite gut is one of the better studied examples, and molecular tools are improving our ability to resolve the taxonomy of the complex relationships among termite gut symbionts.

The hindguts of termites can be compared to small bioreactors where wood and litter are degraded, with the help of symbiotic microorganisms, to provide nutrients. The hindgut of termites is a structured environment with distinct microhabitats (Brune and Friedrich 2000). The dense gut microbiota includes organisms from the Bacteria, Archaea, eukaryotes, and yeasts. These diverse organisms do not occur randomly within the gut but may be suspended in the gut contents, located within or on the surface of flagellates, or attached to the gut wall. The identity, exact number, and location of most is inadequately known because these organisms are difficult to culture. Molecular tools are providing significant new information. The spirochaetes, which account for as many as 50% of the organisms present in some termites, are a distinct phylum within the bacterial domain, but relatively little is known about them. One molecular analysis of spirochaetes in the termite Reticulitermes flavipes suggested there are at least 21 previously unknown species of Treponema (Lilburn et al. 1999). The authors concluded that the long-recognized and striking morphological diversity of termite gut spirochaetes is paralleled by their genetic diversity, which could reflect substantial physiological diversity (Lilburn et al. 1999).

Omnivorous cockroaches also have gut microbial communities, but the associations are less interdependent than those of termites. As in termites, the gut microbial communities in cockroaches anaerobically degrade plant polymers and include hydrogen-consuming bacteria, especially methanogens. The densities of these microorganisms can be enormous; for example, 5 x 1012 bacteria per ml were found in the hindgut of the cockroach Periplaneta americana (Cazemier et al. 1997).

Antlions (Myrmeleontidae) suck out the body fluids of their prey after first paralyzing them with a toxin produced by salivary gland secretions produced by bacteria located in the salivary glands. The paralyzing toxin produced by these bacterial endosymbionts is a homolog of GroEL, a heat-shock protein that functions as a molecular chaperone in E. coli (Yoshida et al. 2001). In the antlion, the GroEL protein may act on receptors in prey insects to induce paralysis. The antlion symbionts perhaps evolved this nonchaperone function to establish a mutually beneficial antlion-bacterium relationship. Yoshida et al. (2001) speculated that insecticidal proteins may be produced by other endosymbionts to help additional fluid-feeding predatory insects.

Tsetse flies (Glossinidae) are vectors of African sleeping sickness disease in humans and animals. Microorganisms associated with these flies, which are blood feeders, are responsible for nutrients not found in their restricted diet. Different microorganisms have been found in the midgut, hemolymph, fat body, and ovaries. Until molecular techniques were used, their taxonomic status was unresolved (Aksoy 2000). Now we know that at least three different microorganisms are present: the primary (P) symbiont, Wigglesworthia glossinidia, is an intracellular symbiont residing in specialized epithelial cells that form a special U-shaped organ (bacteriome) in the anterior gut. The secondary gut symbiont, Sodalis glossinidius, is present in midgut cells. The third, Wolbachia, is found in reproductive tissues. Tsetse females are viviparous, retaining each egg within the uterus where it hatches. The larva matures there and is born as a fully developed third-instar larva. During its intrauterine life, the larva receives nutrients and both of the gut symbionts from its mother via milk-gland secretions; the Wolbachia are transmitted transovarially. Efforts to eliminate tsetse symbionts with antibiotics result in retarded growth and a decrease in egg production. Because it is difficult to eliminate only one symbiont at a time, it is difficult to decipher the role each plays. However, the gut symbionts supply B-complex vitamins, and Sodalis also produces a chitinase, which appears responsible for increasing the susceptibility of its host to the sleeping sickness trypanosome (Aksoy 2000). Analysis of the Wigglesworthia and Sodalis genomes indicates that they each form a distinct lineage in the Proteobacteria. Molecular analyses suggest that a tsetse ancestor was infected with a Wigglesworthia and from this ancestral pair evolved the tsetse species and Wigglesworthia strains existing today. No evidence was found for horizontal transfer of Wigglesworthia symbionts between tsetse species. Sodalis infections might represent recent independent acquisition by each tsetse species or multiple horizontal transfers between tsetse species.

Among the best-studied endosymbionts of insects is Buchnera aphidicola, a bacteriocyte-associated endosymbiont of aphids (Baumann et al. 1997, Douglas 1998, Moran and Baumann 2000). Its complete genome has been sequenced (Shigenobu et al. 2000). Buchnera is found in huge cells (bacteriocytes) in most of the 4400 aphid species, supplying the aphids with essential amino acids. In return, Buchnera is given a stable and nutrient-rich environment. Aphids become sterile or die if their symbionts are eliminated. The aphid-Buchnera relationship has been stable for up to 250 million years, and about 9% of the Buchnera genome is devoted to producing essential amino acids for the aphid. Genes for nonessential amino acids are absent in Buchnera, and this symbiont depends on its aphid host for these, making Buchnera and the aphid codependent.

Analyses of different aphid species and their Buchnera symbionts indicate that vertical transmission of the symbionts has occurred from the time of the common ancestor of aphids, approximately 150 to 250 million years ago (Moran and Baumann 2000). Thus, there is "phylogenetic congruence with hosts, implying co-speciation," and there is no evidence of horizontal transfer, even within a single aphid species (Moran and Baumann 2000). In many Buchnera lineages, genes involved in tryptophan and leucine biosynthesis are present on plasmids rather than in the Buchnera genome. The location of these genes on plasmids allows increased gene expression and, thus, increased benefit to their aphid hosts. The number of copies of the plasmids appears to vary across Buchnera in different aphid lineages, perhaps reflecting coordinated, adaptive adjustment to the nutritional needs of the different aphid hosts. The genome of Buchnera is unusual when compared to the free-living bacterium E. coli. First, the sequences are very AT-biased (about 28% GC). Second, DNA sequences evolve faster in Buchnera than in free-living relatives. Third, the genome of Buchnera (from A. pisum) is reduced to about 650 kb, which is about one-seventh of the genome size of E. coli. Buchnera appears to contain only a subset of about 600 of the 4500 genes present in an E. coli-like ancestor.

Remarkably, it appears that each Buchnera contains 50 to 200 chromosomes, with the number of copies varying with the life-cycle stage of the host. Chromosome amplification may be used to vary the contribution of the symbiont to its host's nutrition (Komaki and Ishikawa 1999, 2000). The amplification of chromosome copy number to 200 copies/cell is very unusual in the microbial world; E. coli typically has one or two chromosomes per cell. The dramatic reduction in genome size of Buchnera and the extraordinary increase in genome copy number make this intracellular symbiont resemble eukaryotic cell organelles such as mitochondria and chloroplasts—which are evolutionary descendants of symbiotic bacteria (Komaki and Ishikawa 2000). Buchnera resemble these organelles also in that they are transmitted maternally between aphid generations.

A less intimate relationship between microbial genomes and insects is represented by the relationship between Enterobacter agglomerans, found in the gut of the apple maggot Rhagoletis pomonella (Lauzon et al. 2000). Enterobacteriaceae are the most common microorganisms associated with the apple maggot in the gut and female reproductive organs, and there are suggestions the flies use the bacteria for some vital function(s) (Lauzon et al. 1998). In addition to E. agglomerans, Klebsiella oxytoca is found in the gut of R. pomonella, and both are most abundant in the esophageal bulb, crop, and midgut. These bacteria are found on host plants and other substrates in the environment. It appears that the bacteria provide usable nitrogen for R. pomonella and other tephritids by degrading purines and purine derivatives, making them facultative symbionts. The relationship between the Enterobacter and Klebsiella species is probably complex. Figure 4.3 illustrates the biofilm of E. agglomerans and Klebsiella in an adult R. pomonella midgut. A biofilm is a complex, structured community of microbes attached to surfaces. Microbial biofilms function as a cooperative consortium in a complex and coordinated manner (Davey and O'Toole 2000). The role of this biofilm in R. pomonella is under study (Lauzon et al. 1998).

Habitant Enterobacteriaceae
Figure 4.3. This scanning electron micrograph shows a biofilm of Enterobacter agglomerans and Klebsiella species in the midgut of the apple maggot Rhagoletis pomonella. (Photo kindly provided by C. R. Lauzon.)

4.13. Insect Development

Much of what we know about the genetics of development in insects has been learned by studying Drosophila melanogaster (Lawrence 1992, Wilkins 1993, Powell 1997, Otto 2000), although that is beginning to change (Klingler 1994). Extensive analyses of insect development have become feasible with the tools of molecular genetics, and thousands of papers have been published on the molecular genetics of development in D. melanogaster. Review articles and books have been published on this rapidly advancing field (Lawrence 1992, Wilkins 1993). A complete discussion of development is beyond the scope of this chapter. However, the following provides a brief outline of D. melanogaster embryonic development that will be useful in understanding sex determination, behavior, and P element-mediated transformation (Chapters 9, 10, and 11).

4.13.1. Oocyte Formation in D. melanogaster

A substantial amount of development of the insect embryo is determined in the oocyte, before oocyte (n) and sperm (n) pronuclei fuse to form an (2n) embryo. Oocyte formation in D. melanogaster is complex, involving both somatic and germ-line cells. The ovaries contain oocytes, which are formed from the pole cells, but the cells that surround each egg chamber and make up the walls of the egg chambers are derived from mesoderm (somatic tissues). The pro-oocyte arises in a set of cell divisions within the ovary from an oogonial stem cell. Each oogonial stem cell divides to give a daughter stem cell and a cystoblast cell. The cystoblast cell gives rise to a set of 16 sister cells in four mitotic divisions, which provides a cyst. One of these 16 cells becomes the pro-oocyte, and eventually the oocyte, while its 15 sister cells become nurse cells whose function is to synthesize materials to supply the growing oocyte. The 16-cell cyst, surrounded by a layer of somatic cells, is termed the egg chamber. The final stages of egg chamber development involve covering the cyst with a monolayer of pre-follicle cells, which are somatic in origin. These 80 somatic cells divide an additional four times to give 1200 follicle cells which cover each cyst.

Initially Drosophila oocytes and nurse cells are roughly the same size, but increase in volume by approximately 40-fold when vitellogenin begins to accumulate about halfway through development of the oocyte. Some vitellogenin is derived from the follicle cells, but most is produced in the fat body and transported to the ovary (Raikhel and Dhadialla 1992). The later stages of oocyte development involve very rapid growth, with the oocyte increasing in volume 1500-fold. While the oocyte is increasing in size, the nurse cells are decreasing because their contents are being deposited in the oocyte. Nurse cells, derived from the germ line, are polyploid, containing 512 and 1024 times the haploid DNA content. These polyploid nurse cells synthesize proteins, ribosomes, and mRNAs. These products, and mitochondria, are transferred to the oocyte by intercellular channels. Thus, the oocyte contains products produced by the mother, which means that initial development in the oocyte is highly dependent upon the genome of the mother (= maternal effects). Finally, the vitelline membrane and the chorion are secreted around the oocyte by follicle cells, and the oocyte enters metaphase of meiosis I. Follicle cells are polyploid, secreting the vitelline membrane of the oocyte and the chorion. The oocyte remains arrested at metaphase of meiosis I until after fertilization.

The oocyte increases in total volume during its development by approximately 90,000fold. Oogenesis is a complex developmental pathway that is estimated to require the function of 70 to 80% of all genes in the Drosophila genome, although the great majority are expressed during other stages of development as well. Only about 75 genes are expressed exclusively during oogenesis (Perrimon et al. 1986). The egg of D. melanogaster is rich in stored RNA, including rRNA and mRNA. The bulk of the maternally produced, stored mRNA is derived from transcription of nurse cell nuclei during egg chamber growth, but some mRNA may be derived from the oocyte nucleus itself, which is active briefly about halfway through development. The total amount of mRNA in the oocyte is equal to about 10% of the single-copy DNA of the Drosophila genome and corresponds to approximately 8000 distinct protein coding sequences. Most of the mRNA codes for proteins that are required early in embryogenesis, including proteins such as tubulins and histones. Products from a few maternal genes continue to affect development in D. melanogaster during the larval stage.

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