Possible functions of the bacterial community Nitrogen fixation and cycling

Obtaining sufficient nitrogen is a major challenge for insects utilizing diets with high car-bon:nitrogen ratios . Termites were studied in this context and are known to compensate for their skewed dietary C:N balance by nurturing a diazotrophic (nitrogen fixing) gut microbiota and acquiring much of their nitrogen directly from the atmosphere (Breznak et al ., 1973; Breznak, 1982) . A number of reports suggest that nitrogen provisioning bacteria may be more ubiquitous among arthropods than previously suspected (reviewed by Nardi et al ., 2002; see also Kneip et al ., 2007) .

Many of the bacteria resident in the digestive system of fruit flies, notably species of Klebsiella (K. oxytoca and K. pneumoniae) and Enterobacter (E. agglomerans and E. cloacae), are diazotrophic As the diet of flies is poor in protein through all stages of development (Yuval and Hendrichs, 2000; Yuval et al ., 1998), several attempts have been made to test the hypothesis that these enterobacteria contribute to the nutritional ecology of these flies by fixing atmospheric nitrogen while resident in the fly gut. Howard and coworkers (1985) found no evidence in favor of nitrogenase activity within larvae and adults of a laboratory strain of R. pomonella. However, nitrogen fixation was detected in B. tryoni after the flies were allowed to feed on cultured Klebsiella and Enterobacter bacteria (Murphy et al , 1988) . Recently, we demonstrated that nitrogen fixation occurs in vivo in wild medflies and results from the activity of stable and dominant populations of diazotrophic Entero-bacteriaceae located in the gut of these flies (Behar et al., 2005). Nitrogen fixation was estimated to proceed at a rate that could provide as much as 6 ]g protein/fly/day (Behar et al ., 2005)—a significant proportion of the medfly's daily nitrogen requirement (Galun et al ., 1985). Furthermore, nitrogen fixation may also be taking place within medfly larvae . Behar et al (2008a) found that a gene responsible for fixing atmospheric nitrogen (nif H) is expressed within the larval gut As larvae experience a high C:N environment within the fruit and protein is essential for larval growth (Yuval and Hendrichs, 2000, Kaspi et al , 2002), nitrogen fixation could contribute to larval nitrogen diet by supplying necessary nitrogenous compounds As most of these diazotrophic Enterobacteriaceae also constitute the dominant intestinal populations of several other tephritids (Table 11 1), nitrogen fixation may be more common among tephritid fruit flies than previously accounted for

Bacteria may further contribute to the nitrogen budget of their hosts by recycling nitrogenous waste products (namely uric acid and ammonia) back into usable compounds Termites as well as cockroaches utilize the uricolytic capabilities of their associated symbionts in order to conserve nitrogen (Potrikus and Breznak 1980b, 1980a, 1981; Cochran, 1985) . It has been suggested that some enterobacterial populations (e.g., Enterobacter spp .) produce uricase, an enzyme degrading uric acid into allantoin, which is later degraded into urea (as shown in Rhagoletis pomonella by Lauzon et al ., 2000), and urease, an enzyme degrading urea into ammonia (e . g., by Providencia stuartii, K. oxytoca, and E. gergoviae; Zinder and Dworkin, 2000) .

Bird feces, a major source of protein for fruit flies, also serve as a major reservoir of bacteria, such as Enterobacter spp Prokopy and coworkers (1993) demonstrated that attraction to bird feces ceases with the addition of antibiotics Therefore, it may be that fruit flies horizontally acquire bacteria to degrade uric acid in the adult fly midgut to a usable form (Lauzon et al 2000) .

The sensor-receptor complex regulating ammonia levels in bacterial cells may constitute a component of the regulatory process of the nitrogen fixation regulatory proteins in Klebsiella pneumoniae (Zhang et al ., 2001) . This might suggest that the combination of Entero-bacteriaceae with different enzymes and sensors involved in nitrogen fixation/uric-acid cycle in the fly's gut may greatly facilitate rapid adaptive responses to fluctuating levels of nitrogen availability and couple the fly's nitrogen metabolism with bacterial activities

As adults, medflies and other fruit flies are anautogenous and need to acquire protein in order to fulfill their reproductive potential (Drew and Yuval, 2000) . A steady supply of fixed and/or recycled nitrogen, generated by internal symbionts, could provide the protein needed to facilitate egg production in females and high sexual activity in males, particularly under poor dietary conditions . Flies could benefit directly from such activity, by assimilating the ammonia generated in these processes and using it for amino acid synthesis, or indirectly by simply digesting their gut bacteria The exact contribution of intestinal bacteria to the nitrogen budget of the flies still awaits clarification

Carbon metabolism

Medfly larvae are known to produce several proteolytic enzymes as well as carbohydrases during their development (Silva et al ., 2006) and hence do not seem to depend on bacteria for the digestion of protein and simple sugars—as suggested for other fruit flies (Hagen, 1966; see also Fitt and O'Brien, 1985). However, their ability to degrade polysaccharides seems to be limited (see Silva et al ., 2006). Bacteria, on the other hand, are excellent at degrading polysaccharides such as cellulose and pectin—an attribute that was previously shown to accompany larval development in Rhagoletis pomonella (Rossiter et al ., 1983) .

Pectinolytic Enterobacteriaceae, mainly K. oxytoca and Pectobacterium spp ., were indeed found to comprise the dominant gut bacterial populations during the larval stages of the medfly (Behar et al , 2008a) Because these larvae need to rapidly acquire sufficient nutrients in order to graduate to the next ontogenetic stage (Kaspi et al ., 2002), bacteria-assisted pectinolysis within the fruit may contribute to the larval carbon diet by providing an additional carbon source of readily metabolizable sugars for the growing larvae Additionally, by macerating the fruit cell walls, pectinolysis may also provide the larvae a more habitable environment, and assist in movement and emergence from the fruit As viscosity or frictional forces decrease in a rotting fruit, so would the energetic cost of movements . Very few studies have addressed this subject linked to biomechanics (e .g., Podolsky, 1994) . Furthermore, the ample supply of readily metabolizable carbohydrates produced by pectin degradation may also fuel the energy-demanding nitrogen-fixation process presumed to occur within the larvae

Adult flies, on the other hand, obtain readily available sugars from fruit juices, honey-dew, and nectar (Tsitsipis, 1989; Drew and Yuval, 2000) and therefore may not need a large pectinolytic microbiota The observed decline in the titer of pectinolytic bacteria in the gut during the adult stage (Behar et al , 2008a) is consistent with this claim More studies are needed to critically examine the contribution of the pectinolytic enterobacterial community to larval development

Communication

Foraging fruit flies have long been known to be attracted to volatiles originating from bacterial catabolism of substrates containing protein (reviewed by Drew and Lloyd, 1991; Lauzon, 2003). Although ammonia seems to be a universal attractant, other volatiles of bacterial origin have been shown to attract fruit flies (Drew and Faye, 1988; Robacker and Flath, 1995; Robacker and Bartelt, 1997; Robacker and Lauzon, 2002; Epsky et al ., 1998) . Such behavior probably represents an adaptation for locating protein sources in the field, and seems not to be directed exclusively to bacteria associated with fruit flies (Robacker et al ., 1998; Lauzon, 2003) Thus, bacteria contribute to the chemical ecology of pest tephritids by affecting their spatial distribution and highlighting resource rich spots

Another aspect of fruit fly bacteria interactions involves volatiles of bacterial origin that serve as semiochemicals affecting adult behavior. Some fruit flies are attracted to bacteria isolated from oviposition wounds and held free of a medium (MacCollom et al ., 1992, 1994) . Accordingly, volatiles produced by bacteria may represent more than just a promise for a protein meal, and mediate more complex behaviors affecting fitness That bacteria or their metabolites act in communication is well documented from a variety of insects: hindgut bacteria were shown to enhance social interactions by contributing to pheromone synthesis in the desert locust, Schistocerca gregaria (Enterobacter, Pantoea, Klebsiella spp.; Dillon et al ., 2000, 2002), and are suspected to do so also in the cockroach, Periplaneta americana (Cruden and Markovetz, 1987) Aggregated oviposition was demonstrated to depend on olfactory cues derived from bacteria deposited with the eggs in onion maggot flies (Delia antiqua) (Judd and Borden, 1992) . In this case Pectobacterium carotovorum may be the bacterium involved (see Judd and Burden, 1992, and references therein) . Similar behavior was also recorded in the house fly, Musca domestica, and was attributed to the proliferation of maternally derived Klebsiella oxytoca bacteria on the eggs and oviposition substrate (Lam et al , 2007) Despite the use of host marking pheromones that deter other females from ovipositing in the same fruit, medfly females are also known to occasionally oviposit in an aggregated fashion (Diaz-Fleischer et al ., 2000). Aggregated oviposition may be in the best interest of females, especially when ovipositing into large fruits, because the crowded development of larvae can strongly inhibit the occurrence of pathogenic agents in rotting, decomposing substrates (e .g., Rohlfs et al ., 2005) . However, overcrowding or unsynchro-nized egg hatch could result in strong competition among the larvae Bacteria deposited with the eggs into the host may produce volatile cues that provide arriving gravid females with information on the density and age of eggs already incubating within the fruit Such information, along with pheromonal cues, could allow females to make optimal reproductive decisions

The intestinal microbiota may also affect the fitness of male medflies . Ben-Yosef and coworkers (2008) recently examined the effects of intestinal bacteria on medfly fitness and its relation to diet A significant reduction in sexual competitiveness of males fed with antibiotics while provided with all nutritional requirements was evident. On the other hand, clearing the gut of bacteria did not affect the ability of males fed only on sugar to achieve copulations . These findings are compatible with the work of Niyazi et al . (2004) who demonstrated a mating advantage in probiotically treated sterile males fed with protein One way to explain these results is that intestinal bacteria influence male copulatory success by qualitatively or quantitatively contributing to pheromone production. Protein may be a prerequisite for such activity due to its positive effects on pheromone emission and copulatory success (Papadopoulos et al , 1998; Blay and Yuval, 1997) By synthesizing pheromonal precursors or alternatively modifying existing molecules produced by the males, hindgut bacteria may provide the females with information about the male's health and nutritional status These may be important parameters influencing mate choice in Lek mating systems, such as that of the medfly (Field et al , 2002), where males provide the females with nothing but their genes

Defense against pathogens

Relatively little information exists in the area of tephritid pathology even though fruit flies consume and are exposed to insect pathogens such as cricket paralysis virus (Manousis and Moore, 1987), reoviruses and reo-like viruses (Plus et al ., 1981a, 1981b; Lauzon, unpublished), Wolbachia (Kittayapong et al ., 2000; Riegler and Stauffer, 2002; Selivon et al ., 2002; Zabalou et al ., 2004; Rocha et al ., 2005), microsporidia (Fujii and Tamashiro, 1972), and Ser-ratia marcescens (Steinhaus, 1959; Grimont and Grimont, 1978; Lauzon et al ., 2002) . Isolates of the latter were lethal to Rhagoletis pomonella (Lauzon et al , 2003)

Moreover, as mentioned earlier, some of the Pseudomonas strains forming the minor yet common and stable community in the medfly's gut are pathogenic . When higher than natural levels of Pseudomonas aeruginosa were orally introduced to the medfly's digestive system they reduced host longevity, while ingesting higher levels of the medfly's gut enterobacterial community improved host longevity (Behar, unpublished) These results suggest that at least part of the Pseudomonas community present in the gut cause damage to its medfly host when occurring at, or reaching, high densities

The gut microbiota of silkworm larvae and locusts, among them the enterobacterium Pantoea agglomerans, provide a buffering action to help prevent the proliferation of pathogens (reviewed by Dillon and Dillon, 2004) Because the medfly's gut enterobacterial community dominantly establishes during the adult stage within the medfly's gut (Behar et al ., 2008a) and contributes to their host longevity (as mentioned above), we postulate that by preventing the establishment and proliferation of harmful bacteria, the Enterobacteriaceae community may play a similar role in the medfly's gut Thus, by keeping the Pseudomonas community in check, the dominant establishment of the Enterobacteriaceae community within the medfly's gut contributes to the fly's longevity, acting as a physical barrier against deleterious (foreign and indigenous) bacteria More information needs to be acquired on pathogens because mass rearing programs must include plans to control and/or eliminate these microorganisms within the rearing facility During studies that showed that a diet for medflies that includes beneficial symbionts improved the gut of irradiated flies used in the sterile insect technique (Lauzon and Potter, 2008) and their mating performance (Niyazi et al ., 2004), Lauzon also found that bacteria that typically resided in the facility diet were eliminated or did not become established when the beneficial symbionts were present (unpublished) This dynamic decreases the need for antimicrobial use in mass rearing and may reflect a protective mechanism exerted by beneficial symbionts for fruit flies

Phytopathology

Larval development within the fruit is accompanied by a rapid deterioration of the fruit pulp During oviposition, fly-associated Enterobacteriaceae, mainly Citrobacter freundii, Klebsiella oxytoca, Pantoea spp., and Pectobacterium cypripedii (Table 11 2), are transmitted to the fruit along with the eggs and subsequently proliferate within it. Combined with feeding activity of larvae, these bacteria accelerate fruit decay (Behar et al , 2008a) Some species of the enterobacterial community in the medfly's gut, such as Pantoea spp and Pectobacterium cypripedii, are known phytopathogens due to their ability to degrade pectin

(Zinder and Dworkin, 2000) . Strains of K. oxytoca and Pectobacterium spp . isolated from the medfly's gut caused decay in potatoes (Behar, unpublished)—a known test for pectino-lytic activity by bacteria (Page et al , 2001) In this capacity medflies (and other tephritids) may act as vectors of phytopathogenic bacteria Because more than 300 species of fruit are confirmed as hosts for ovipositing medfly females, this mechanism may have a major agricultural significance

Interactions at the ecosystem level

The host plant has been identified as a mediator between fruit flies and bacteria (Drew and Lloyd, 1987) This concept can be extended by looking at the fruit within which larvae develop as a "microbial hub ." Under natural conditions, oviposition hosts can be shared by conspecifics and, although the medfly usually is the first to attack the fruit and precipitate its decay, by other species In nature, these other species are often other flies (mainly Dro-sophilids), beetles, and the attendant community of natural enemies, who rapidly proliferate within the decaying fruit A number of interesting questions arise: what brings about or inhibits sharing of resources (the decaying fruit) with other species? Is there a gain of fitness, and are microorganisms involved in this gain (Rohlfs and Hoffmeister, 2003)? Natural bacterial populations of Drosophila may be quite different from those of the medfly (Cox and Gilmore, 2007; Corby-Harris et al ., 2007). If this is true for populations sharing larval feeding sites, how is this selectivity achieved, and can members of the community of one insect species colonize the other? Finally, the intriguing possibility of lateral gene transfer occurring in this setting arises Such a process could contribute to the evolution of the interactions between the insects and their microbial partners, as well as to the diversity and fitness of the bacterial populations involved

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