Symbiotic Microorganisms are the components of the microbiota of an insect that contribute to insect survival, growth, or fecundity. They are borne by an estimated 10% of all insect species, and are located in the insect gut or tissues, often restricted to specialized cells called bacteriocytes. Most of the microorganisms are rare or unknown apart from the insect partner, and many have not been cultured in vitro. Historically, the microbiology of insects has been little studied and, until recently, most of the information available on symbiotic microorganisms has been derived from microscopic analysis of the insect regarding the morphology of the microorganisms, their location in the insect body, and their mode of transmission between insects. The advent of molecular techniques has transformed our understanding of symbiotic microorganisms, allowing the taxonomic identification of microorganisms and the elucidation of the molecular basis of their function. Because of the insects’ dependence on their symbiotic microorganisms, these associations are of great potential value as a novel approach to insect-pest management.
I. DIVERSITY OF SYMBIOTIC MICROORGANISMS IN INSECTS
A. Distribution of symbiotic microorganisms across the microbial kingdoms Symbiotic microorganisms include members of all microbial kingdoms (Table 54.1)—various Eubacteria, methanogens (Archaea), and protists and fungi (Eukaryota). The Eubacteria, especially members of the _-Protobacteria, are widely represented both in the guts and cells of insects, but methanogens and protists occur in the strictly anaerobic portions of the guts of certain insects. Yeasts have been reported in the gut lumen, cells, and haemocoel (body cavity) of some species. (Basidiomycete fungi are also cultivated in the nests of some insects, e.g., fungus-gardening termites and leaf-cutting ants, but these ectosymbioses are not considered here.) B. Symbiotic microorganisms in insect guts Most insects have a substantial gut microbiota, although there are wide differences among insect taxa and among regions of the gut. Much of the literature gives misleading estimates of the microbial diversity in insect guts because the techniques commonly used are based on culturable forms, which account for only 0.1–10% of the total microbiota. An additional complication to the study of symbiotic microorganisms in insect guts is that many or all members of the microbiota are either transient (i.e., passing through the gut with the unidirectional passage of food), or commensal (i.e., resident for extended periods, but of no discernible advantage to the insect). Detailed experimental study is required to identify which, if any, of the resident gut microbiota are symbiotic microorganisms (i.e., beneficial to the insect). The microbiology of termite guts has been studied extensively. The greatest density of microorganisms is in the anoxic proximal portion of the hindgut, known as the paunch. In all termites, this region harbors bacteria, at 109–1010 cells/ml gut volume. All the bacteria are facultative or obligate anaerobes. They comprise methanogens, spirochaetes (pillotinas, e.g., Hollandia, Pillotina, Diplocalyx, and Clevelandina, and many other unidentified forms), and other eubacteria, including species of Enterobacter, Bacteroides, Bacillus, Citrobacter, Streptococcus, and Staphylococcus. The lower termites in addition have obligately anaerobic, flagellate protists of the orders Hypermastigida and Trichomonadida and Oxymonadida at densities of up to 107 cells/ml (Fig. 54.1).
Approximately 400 species of these protists have been reported and a few, including Trichomitopsis termopsidus and Trichonympha sphaerica (both from the termite Zootermopsis) have been brought into axenic culture. Higher termites lack these protists. Apart from the termites, most studies have concerned the gut microbiota of the few insect species that are routinely reared in laboratories. As examples, the American cockroach Periplaneta americana bears both obligately anaerobic bacteria (e.g., Clostridium and Fusobacterium) at densities of ~1010 cells/ml and facultative anaerobes (e.g., Klebsiella, Yersinia, and Bacteroides) at ~108 cells/ml; the locust Schistocerca gregaria has exclusively facultative anaerobes, usually at considerably lower densities than the cockroach; and the blood-feeding reduviid bug Rhodnius prolixus has a diversity of bacteria, including Pseudomonas, Streptococcus, Corynebacterium, and various actinomycetes (and not a single gut symbiont, the actinomycete Nocardia rhodnii, as claimed in much of the early literature). The composition of microorganisms in the guts of insects can vary widely with environmental circumstance. The microbiota may change when insects are transferred to laboratory rearings, and specific differences in the microbiota of insects in different laboratories or under different temperature or dietary regimes have been described. C. Symbiotic microorganisms in insect tissues and cells Many symbiotic microorganisms are located in insect cells, where they may be well protected from the hemolymph-based defense system of the insect. In any single insect, the cells bearing intracellular symbiotic microorganisms are usually of a single morphological form (e.g., see Fig. 54.2) and location in the insect body (see Table 54.1).
They are called bacteriocytes or mycetocytes, and their sole function appears to be the housing of the microorganisms. The incidence of intracellular microorganisms in insects and, where known, taxonomic information on the microorganisms are summarized in Table 54.1. Most of the microorganisms are Eubacteria but, because they have not been brought into axenic culture, taxonomic information is available only for those forms whose 16S rRNA has been sequenced. Many of the bacteria are members of the _-Protobacteria, and the microorganisms in three taxonomically disparate insect groups, aphids, tsetse flies, and ants, are particularly closely related. The bacteria in aphids and tsetse flies are known as Buchnera spp. and Wigglesworthia spp., respectively, in recognition of the research of entomologists Buchner and Wigglesworth on these systems. Other intracellular bacteria in bacteriocytes include members of the _-Protobacteria in mealybugs and flavobacteria in cockroaches. The bacteria in several insect groups, including the Anoplura and Mallophaga (the sucking and chewing lice, respectively) and various Heteroptera, have not been studied at the molecular level (see Table 54.1). Some insects bear microorganisms whose location (intracellular vs. extracellular) is variable. For example, delphacid planthoppers and hormaphidine aphids lack intracellular bacteria (unlike other planthoppers and aphids), and bear pyrenomycete yeasts in and between fat body cells. Some species of aphid and tsetse fly have secondary symbionts, in addition to Buchnera and Wigglesworthia, respectively. The secondary symbionts are variably located in cells and the haemocoel, and of uncertain significance to the insect.
II. FUNCTION OF SYMBIOTIC MICROORGANISMS
A. Symbiotic microorganisms as a source of novel metabolic capabilities Symbiotic microorganisms are widely believed to contribute to the nutrition of insects. This was first deduced from the distribution of the associations among insects. In general, the microorganims are restricted to insects living on nutritionally poor or unbalanced diets. They are widespread or universal among insects feeding through the lifecycle on the phloem and xylem sap of plants, deficient in essential amino acids; vertebrate blood, deficient in B vitamins; and wood, which is composed principally of lignocellulose and is deficient in many essential nutrients for insects. The implication is that the symbiotic microorganisms variously degrade cellulose and synthesize essential amino acids and vitamins. They have also been implicated in the synthesis of sterols, which insects and other arthropods cannot synthesize de novo. The symbiotic microorganisms can be considered to be a source of biochemically and genetically complex metabolic capabilities that the insect lacks. They have also been described as microbial brokers, mediating insect utilization of blood, plant sap, and wood. B. Contribution of symbiotic microorganisms to the nitrogen nutrition of insects Three routes have been identified by which symbiotic microorganisms contribute to the nitrogen nutrition of insects, the fixation of N2, nitrogen recycling, and, the synthesis of essential amino acids. Symbiotic microorganisms in the gut of some insects fix N2 at appreciable rates; no intracellular N2-fixing bacteria in bacteriocytes have been described. In particular, many termite species derive significant supplementary nitrogen from N2-fixing bacteria (e.g., Enterobacter agglomerans and Citrobacter freundii) in the anoxic portion of their hindgut. The N2 fixation rate varies widely among termite species, from _0.2g N fixed/g insect weight/day in Labiotermes sp. and Cubitermes sp. to _6g N/g/day in Nasutitermes species, and is also influenced by environmental conditions, including the concentration of combined nitrogen in the diet. Nitrogen recycling refers to the microbial consumption of nitrogenous waste products of insects and the synthesis of compounds (e.g., essential amino acids) of nutritional value to the insect, which are then translocated back to the animal. Microbial utilization of insect-derived uric acid or ammonia has been demonstrated in several systems, including cockroaches, planthoppers, aphids, and termites.
For example, various bacteria, including Streptococcus, Bacteroides, and Citrobacter species, in the hindgut of the termite Reticulotermes flavipes degrade uric acid anaerobically to ammonia, carbon dioxide, and acetic acid. Experiments using 14C and 15N-labeled uric acid confirmed that uric acid is degraded by the hindgut microbiota in the insect and nitrogen is subsequently assimilated by the insect tissues, in the insect. Microbial provision of essential amino acids to the insect has been studied systematically in the symbiosis between aphids and the intracellular bacteria Buchnera. The core evidence is nutritional, and arises from the development of chemically defined diets consisting of sucrose, amino acids, vitamins, and minerals, on which aphids can be reared. Dietary studies in which the 20 amino acids of proteins are individually omitted have revealed that many aphids have no specific requirement for most or all the amino acids that are normally dietary essentials for animals, but that aphids experimentally deprived of Buchnera by antibiotic treatment require all the essential amino acids. The implication, that the insect derives essential amino acids from Buchnera, is supported by radiotracer studies demonstrating the synthesis de novo of various essential amino acids by aphids bearing Buchnera (Fig. 54.3). The analysis of the plasmid profiles in Buchnera has revealed molecular support for the role of these bacteria in the amino acid nutrition of aphids. Buchnera in many aphids, including all members of the family Aphididae studied to date, bear two multicopy plasmids on which genes for the biosynthesis of tryptophan and leucine are amplified. Figure 54.4 shows the genetic organization of these plasmids in the Buchnera from which they were first described. The synthesis of essential amino acid by intracellular bacteria has not been studied systematically in any insects apart from aphids. There is, however, a strong but unproven supposition that many insects, especially phloem-feeding Homoptera (e.g., whitefly and psyllids) and cockroaches, derive these nutrients from their symbiotic microorganisms. If confirmed, the essential amino acid provisioning has evolved independently in different bacterial groups. C. Vitamin synthesis by symbiotic micro-organisms The microbial provision of B vitamins has been proposed for the diverse insect taxa that feed on vertebrate blood and bear microorganisms in either the gut or bacteriocytes.
The experimental basis for this role is exclusively nutritional, based on insect performance. Triatomid bugs deprived of their gut microbiota die as larvae, but this developmental arrest is alleviated by injection of B vitamins into the insect or vitamin supplementation of the diet. Similarly, the larvae of the louse Pediculus lacking symbiotic bacteria suffer high mortality, unless the blood diet is supplemented with nicotinic acid, pantothenic acid, and biotin, and B vitamins have been reported to partially restore the fecundity of tsetse fly from which the bacteria are eliminated. Other insects feeding on nutritionally poor diets, (e.g., cockroaches and timber beetles) may also derive vitamins from symbiotic microorganisms. The anobiid beetle Stegobium paniceum is independent of several B vitamins, riboflavin, nicotinic acid, pyridoxine, and pantothenic acid, but insects from which the yeasts are eliminated require these vitamins for normal development. D. Sterol synthesis by symbiotic microorganisms Yeasts may contribute to the sterol nutrition of insects. Despite some claims in the literature, for example, that aphids derive sterols from their symbiotic bacteria Buchnera, bacterial provision of these nutrients is most improbable because the Eubacteria are not capable of substantial sterol synthesis. Yeasts have been implicated in the sterol nutrition of some planthoppers and timber beetles. For example, when the yeast population in the planthopper Laodelphax striatellus is depleted, many of the insects die during the final molt to adulthood, but mortality was reduced from 94% to 40% by injecting the insects with either cholesterol or the plant sterol sistosterol. E. Cellulose degradation Many insects feeding on fiber-rich plant material, especially wood, have substantial gut microbiota. For many years, these insects have been assumed to be strictly comparable to vertebrate herbivores in which microorganisms mediate cellulose degradation. This can be illustrated by microbe-mediated cellulolysis in lower termites. Protists in the hindgut of lower termites (see Section I.B) can be eliminated by incubating the insects at elevated oxygen tensions, and these protist-free insects, commonly termed defaunated termites, cannot survive on high, cellulose diets, such as filter paper (Fig. 54.5). The protists degrade cellulose, with carbon dioxide and short-chain fatty acids, especially acetate, as the principal products of fermentation. The acetate is absorbed across the hindgut wall and metabolized as a source of energy by the aerobic tissues of the termite. Cellulase active against crystalline cellulose has been demonstrated in the protist Trichomitopsis and in mixed populations of protists from Coptotermes lacteus. Although the experimental data on lower termites are not in serious doubt, this system cannot be generalized to all insects feeding on high-fiber diets.
It is now recognized that some insects (unlike vertebrates) have intrinsic cellulases, especially endoglucanases and _-glucosidases. For example, the higher termites (which lack protists) do not have cellulolytic microbiota, but instead possess high activities of intrinsic cellulases, especially in the midgut (Table 54.2). The contribution of intrinsic and microbial cellulases to cellulose breakdown has been investigated in relatively few insects other than termites. The locust Schistocerca gregaria has intrinsic cellulases; the scarab beetle Pachnoda marginata uses the celluloytic capability of bacteria in its hindgut; and among the woodroaches (cockroaches that feed on wood), Panesthia cribatus uses intrinsic cellulases and has gut microbiota of noncellulolytic bacteria, whereas Cryptocercus punctulatus has up to 25 species of obligately anaerobic protists that degrade cellulose. There is no evidence that the efficiency of fiber digestion by the insect is influenced by the origin (intrinsic or microbial) of the cellulolytic enzymes. Insects with microbial cellulolysis generally have a high population of methanogenic bacteria. These bacteria act as a sink for hydrogen produced by anaerobically respiring microorganisms, and so promote cellulose degradation.
III. DETERMINANTS OF THE DENSITY OF SYMBIOTIC MICROORGANISMS IN INSECTS A. Gut microorganisms Insect guts are physically unstable environments. The gut lumen is dominated by the unidirectional bulk flow of ingested food and many microbial cells pass directly through the gut (see also Section I.B). Microbial persistence in the gut is promoted by: 1. A higher proliferation rate than the rate of passage of the food (this is probably important in insects with cellulolytic microbiota in enlarged fermentation chambers). 2. The adhesion of microorganisms to the gut wall. 3. The sequestration of microorganisms into outpocketings of the gut (e.g., midgut caeca and Malpighian tubules). A further aspect of the instability of the gut environment is that the cuticle and contents of the foregut and hindgut are lost at each insect molt, such that the microbiota is reestablished de novo multiple times through an insect’s lifespan. The midgut microbiota can persist through insect molts, but is commonly lost at the metamorphosis of holometabolous insects (insects with complete metamorphosis, e.g., flies and beetles). In other respects, however, the midgut of many insects is a hostile environment for microorganisms because it is the principal site of digestive enzymes. B. Intracellular microorganisms The symbiotic microorganisms in the cells and tissues of insects are not generally subject to the frequent disturbance experienced by the foregut and hindgut microbiota (see Section III.A), and their populations are probably regulated by density-dependent processes, mediated by the insect. The regulation of intracellular bacteria Buchnera in aphids has been well studied. In the wingless parthenogenetic morph of aphids, the bacterial population increases in parallel with aphid biomass through larval development (Fig. 54.6), and in adult aphids the bacteria occur at a density of ~107 cells/mg aphid fresh weight, equivalent to 10% of the total insect volume. Regulation occurs at two levels, controls over the bacterial division rate in each bacteriocyte such that they maintain a uniform density, equivalent to 60% of the cytoplasmic volume of bacteriocytes, and controls over the number of bacteriocytes through age-dependent lysis of the bacteriocytes and all enclosed bacteria. Adults of winged female aphids and male aphids tend to have a smaller Buchnera population than the wingless morph and these differences are mediated by both a lower rate of bacteriocyte enlargement and higher incidence of bacteriocyte death during larval development. The pos-sibility that these morph-specific differences are mediated by insect hormones has not been investigated. Reduced bacterial populations in males has also been demonstrated in leafhoppers, weevils, and cockroaches.
IV. TRANSMISSION OF SYMBIOTIC MICROORGANISMS
A. Gut microbiota All members of the gut microbiota are acquired via insect feeding. For many microorganisms, the transfer between insects is haphazard, dependent on the chance ingestion with food, but in many taxa, the insect eggs are provisioned with gut microorganisms. The transmission of some microorganisms is assured by stereotyped feeding responses of the insect. This can be illustrated by the heteropteran bug Coptosoma scutellarum, which bears bacteria in its midgut caeca. A capsule bearing bacteria is deposited alongside each oviposited egg. When the larva hatches, it immediately feeds on the capsule contents and acquires its complement of bacteria (Fig. 54.7), on which its growth and development depend. Insect behavior is also implicated in the transmission of obligately anaerobic microorganisms, especially the cellulolytic protists in certain woodroaches and the lower termites. At each molt of the insect, the oxygen tension in the hindgut increases dramatically and all the protists are killed. In Cryptocercus, the protists initiate sexual reproduction just before each molt and are transformed into oxygen-resistant cysts. They are expelled from the hindgut into the environment, where they persist until ingested by the insect after molting. In contrast, the protists in lower termites rarely reproduce sexually and never encyst, and they are killed at each insect molt. The termites acquire a fresh inoculum of protists by feeding on a drop of hindgut contents from the anus of another colony member, a behavior known as proctodeal trophyllaxis. It has been suggested that the requirement for conspecifics as a source of protists may have been a major selection pressure for the evolution of eusociality in termites. Higher termites, which lack the protists, do not exhibit proctodeal trophyllaxis. B. Transovarial transmission Microorganisms located in insect tissues and cells are generally transmitted from mother to offspring via the unfertilized egg in the female ovary. As a result, the symbiotic microorganisms are present even before fertilization and, potentially, for the entire lifespan of the insect. The timing and anatomical details of transovarial transmission vary widely among insect groups, consistent with these associations having evolved independently on multiple occasions.
In some insects (e.g., aphids), the bacteriocytes are closely apposed to the ovaries and the bacteria have a fleeting extracellular stage in transit from bacteriocyte to ovaries. In other insects, the bacteria have a prolonged extracellular phase, either because they are expelled from bacteriocytes at a distance from the ovaries and migrate to the ovaries (e.g., many species of lice) or because they remain on the egg surface for extended periods (e.g., cockroaches). Varying among insect taxa, the bacteria are phagocytosed directly by the egg, usually at the time of vitellogenesis (e.g., aphids and cockroaches) or taken up by insect cells at the base of each ovariole and inoculated into the posterior pole of the egg just prior to formation of the chorion (the egg shell) and ovulation. C. Vertical transmission and its evolutionary consequences Transovarial transmission and the more sophisticated instances of microbial transmission by egg smearing ensure that each egg is colonized by bacteria from its mother. If vertical transmission persists over many insect generations without cross-infection, the insect and microbial lineages evolve in parallel, and their phylogenies are congruent. Congruent phylogenies have been demonstrated for several insect–microbial symbioses, most notably the aphid–Buchnera association, in which the 16S rRNA sequence phylogeny of Buchnera is completely concordant with morphologybased phylogeny of aphids (Fig. 54.8). On the reasonable assumption that the aphids and Buchnera diversified in synchrony, the date of divergence between Buchnera and its relatives, such as Escherichia coli, has been estimated at 180–250 million years ago. Vertical transmission, especially by the transovarial route, has two characteristics of significance for the evolution of symbiotic microorganisms. First, relatively small numbers of cells are transferred from parent to offspring; that is, the effective population size of the microorganisms is small. Second, the strict maternal inheritance prevents any contact between microbial populations in different insects, precluding recombination. Deleterious mutations are likely to accumulate in these small asexual populations. Supportive evidence comes from studies of sequence evolution in intracellular bacteria. First, among the Buchnera lineages, protein-coding genes have significantly elevated the incidence of nonsynonymous substitutions (i.e., those point mutations that alter the amino acid) and this is not paralleled by an increase in the rate of synonymous substitutions (Fig. 54.9A). Second, vertically transmitted intracellular bacteria have base substitutions in the 16S rRNA gene that tend to destablize the secondary structure of the rRNA molecule (Fig. 54.9B).
V. SYMBIOTIC MICROORGANISMS AND INSECT-PEST MANAGEMENT
Many insects that depend on symbiotic microorganisms for sustained growth and fecundity are pests of agricultural or medical importance. They include crop pests (e.g., aphids, whitefly, grain beetles, and timber beetles) and vectors of animal and human pathogens (e.g., tsetse fly and bedbugs). In addition, certain microbial taxa have been implicated in the vector competence of their insect hosts. The transmission of luteoviruses by aphids is promoted by binding of a protein, the chaparonin GroEL derived from the intracellular bacteria Buchnera, to the surface of the virus particles in the insect body, and the inhibition of trypanosome infection in the tsetse fly by an insect lectin is blocked by high N-acetylglucosamine titers generated by the chitinase activity of a midgut bacterium. The selective disruption of the symbiotic microorganisms in insect pests, and the consequent depression in insect performance, would be of considerable economic value but no commercial approach has been developed. There is also interest in the genetic manipulation of symbiotic microorganisms, for example to reduce insect-vector competence. The intracellular microorganisms are generally perceived as intractable because the methods to culture and transform them and to reintroduce the transformed bacteria into insects have not been developed. Most research has been conducted on blood-feeding insects with gut microbiota. For example, a plasmid of the actinomycete Rhodococcus rhodnii, which inhabits the gut of Rhodnius prolixi, has been genetically modified to bear the plasmid replication origins for both R. rhodnii and E. coli, and sustained infections of Rhodnius have been achieved with the transformed bacteria. This technology is being developed as part of a strategy to reduce the Rhodnius-mediated transmission of the protozoan pathogen that causes Chagas’s disease in humans.
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