Microbial combinatorics: a simplified approach for isolating insecticidal bacteria

Bacteria can control pest insects that damage food crops, vector diseases and defoliate trees. Conventionally, isolation of these bacteria has been from soil and sporadically from dead insects. A simplified approach for isolating insecticidal bacteria from soil using the target insect as the selective agent was employed in this study. Instead of isolating single strains of bacteria from soil and testing each individual strain for insect toxicity, mixtures of bacteria present in each soil sample were tested together directly for toxicity using Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae) as a model insect. Thirty-five soil suspensions or bacterial suspensions of the 40 suspensions tested killed at least one M. sexta larva. All but one bacterial culture isolated from dead larvae and retested for toxicity, killed at least one M. sexta larva. Nineteen bacterial strains isolated from larvae killed in the first test, were identical to the bacteria fed to the retested larvae. Of the 19 strains isolated, 14 were identified by 16S rDNA sequencing as belonging to the Bacillus cereus group including three strains that formed crystals that were identified as B. thuringiensis. Of the three other spore-forming strains, two were identified as psychrotrophic B. weihenstephanensis and the third as Lysinibacillus fusiformis. Two others were identified as Enterococcus faecalis. This approach, microbial combinatorics, reduces the number of insects necessary for toxicity screening and associated time and resources compared to conventional methods that first isolate bacteria and then individually test for toxicity as well as a means of discovery of new pathogens using the insect as the selective agent.     

Kinetics of Colonization of Adult Queensland Fruit Flies (Bactrocera tryoni) by Dinitrogen-Fixing Alimentary Tract Bacteria

The average total population of bacteria remained constant in the alimentary tracts of adult laboratory-raised Queensland fruit flies (Bactrocera tryoni) although the insects had ingested large numbers of live bacteria as part of their diet. The mean number of bacteria (about 13 million) present in the gut of the insects from 12 to 55 days after emergence was not significantly modified when, at 5 days after emergence, the flies were fed antibiotic-resistant bacteria belonging to two species commonly isolated from the gut of field-collected B. tryoni. Flies were fed one marked dinitrogen-fixing strain each of either Klebsiella oxytoca or Enterobacter cloacae, and the gastrointestinal tracts of fed flies were shown to be colonized within 7 days by antibiotic-resistant isolates of K. oxytoca but not E. cloacae. The composition of the microbial population also appeared to be stable in that the distribution and frequency of bacterial taxa among individual flies exhibited similar patterns whether or not the flies had been bacteria fed. Isolates of either E. cloacae or K. oxytoca, constituting 70% of the total numbers, were usually dominant, with oxidative species including pseudomonads forming the balance of the population. Antibiotic-resistant bacteria could be spread from one cage of flies to the adjacent surfaces of a second cage within a few days and had reached a control group several meters distant by 3 weeks. Restriction of marked bacteria to the population of one in five flies sampled from the control group over the next 30 days suggested that the bacterial population in the gut of the insect was susceptible to alteration in the first week after emergence but that thereafter it entered a steady state and was less likely to be perturbed by the introduction of newly encountered strains. All populations sampled, including controls, included at least one isolate of the dinitrogen-fixing family Enterobacteriaceae; many were distinct from the marked strains fed to the flies. Nitrogenase activity detected by the acetylene reduction assay was associated with flies fed dinitrogen-fixing bacteria as well as with control groups given either no supplement or free access to a yeast hydrolysate preparation. Nitrogen fixed from the atmosphere may supplement the nutrition of the alimentary tract microbial population of B. tryoni. Transmission electron microscopy showed that the principal site of bacterial colonization in the abdominal alimentary tract was the lumen of the midgut inside the peritrophic membrane. No intracellular symbionts were seen in the gut tissues nor were bacteria found attached to the cuticular folds of the hindgut. The ultrastructure of the gut resembled that of other fly genera except that the intercellular spaces between rectal epithelial cells were more extensive, suggesting a role for unspecialized epithelium in water and solute uptake in B. tryoni.     

Investigation of the bacterial communities associated with females of Lutzomyia sand fly species from South america

Phlebotomine sand flies are vectors of Leishmania that are acquired by the female sand fly during blood feeding on an infected mammal. Leishmania parasites develop exclusively in the gut lumen during their residence in the insect before transmission to a suitable host during the next blood feed. Female phlebotomine sand flies are blood feeding insects but their life style of visiting plants as well as animals, and the propensity for larvae to feed on detritus including animal faeces means that the insect host and parasite are exposed to a range of microorganisms. Thus, the sand fly microbiota may interact with the developing Leishmania population in the gut. The aim of the study was to investigate and identify the bacterial diversity associated with wild adult female Lutzomyia sand flies from different geographical locations in the New World. The bacterial phylotypes recovered from 16S rRNA gene clone libraries obtained from wild caught adult female Lutzomyia sand flies were estimated from direct band sequencing after denaturing gradient gel electrophoresis of bacterial 16 rRNA gene fragments. These results confirm that the Lutzomyia sand flies contain a limited array of bacterial phylotypes across several divisions. Several potential plant-related bacterial sequences were detected including Erwinia sp. and putative Ralstonia sp. from two sand fly species sampled from 3 geographically separated regions in Brazil. Identification of putative human pathogens also demonstrated the potential for sand flies to act as vectors of bacterial pathogens of medical importance in addition to their role in Leishmania transmission.     

Pathogenic effects of bacteria isolated from larvae of Hylesia metabus Crammer (Lepidoptera: Saturniidae)

Hylesia metabus larvae are susceptible to several pathogens indigenous to the area in which they are found. Some larvae show symptoms characteristic of bacterial infection; they become flaccid and lethargic, and show a marked loss of appetite. We isolated and identified 29 bacterial strains from live, dead and experimentally infected H. metabus larvae, and evaluated their pathogenic activity. The bacteria which caused mortality in the larvae were: Pseudomonas aeruginosa (60-93.3%), Proteus vulgaris (20%), Alcaligenes faecalis, Planococcus sp. and Bacillus megaterium (10%), at doses of 3-4 x 10(7). Although P. aeruginosa is a well-known insect pathogen, this is the first report of its pathogenic activity on H. metabus. The potential risk to humans and low virulence make it unlikely that P. aeruginosa could be used in an augmentative biological control programme. However its natural incidence may be enhanced using parasites and predators of H. metabus as carriers.     

Generation and properties of a luminescent insect pathogen Xenorhabdus nematophilus (Enterobacteriaceae)

Studies on the interaction of the insect pathogenic bacterium, Xenorhabdus nematophilus (Enterobacteriaceae), with its nematode and insect hosts would be greatly assisted if a luminescent phenotype were generated that would allow the detection of viable bacteria in vivo without the necessity for disruption of the cellular interactions. The plasmid, pMGM221, containing the luminescence gene (luxCDABE) of Vibrio harveyi was introduced into different strains (DD136 and 19061) and phases (one and two) of X. nematophilus by triparental mating. For reproducible and efficient conjugation, it was necessary to use older cultures (96-160 h) in the stationary phase of X. nematophilus for mating with relatively small differences (<2-fold) in transconjugant yield for the different strains and phases of X. nematophilus. All transconjugants emitted high levels of light with optimum bioluminescence at 27 degrees C in Luria broth at pH 8.0 containing 20 g/L NaCl; pH, osmolarity, and temperature conditions were similar to those encountered by the bacteria in the hemolymph of the larvae of Galleria mellonella. Plasmids were detected in the transconjugants after 6 months of subculturing the bacteria without antibiotic selection. Aside from light emission, luminescent transconjugants had the same physiological properties as the nonluminescent parental strains, including identical rates of growth, production of exoenzymes, removal from and subsequent emergence into the insect's hemolymph, bacterial-induced hemocyte damage, suppression of prophenoloxidase activation, and the ability to kill G. mellonella larvae. Light-emitting larvae could readily be detected by eye in a dark room, and all bacteria reisolated from dead larvae were luminescent. These properties validate the use of luminescent X. nematophilus not only as a means of following bacterial host interactions, but also as a potential agent to follow the infection and death of the insect population.     

In vitro adhesion of n(2)-fixing enteric bacteria to roots of grasses and cereals

Nitrogen-fixing Klebsiella and Enterobacter strains isolated from several plants were assayed for fimbriae and for adhesion to plant roots in vitro. All eight Klebsiella strains formed type 3 fimbriae, and five strains also formed type 1 fimbriae; all 21 Enterobacter strains had type 1 fimbriae. Three strains of Klebsiella carrying either type 1, type 3, or no fimbriae were used as model organisms in developing an in vitro adhesion test. Adhesion was assayed with bacterial cells labeled with [H]leucine. Fifteen N(2)-fixing strains and the three model strains were compared for adhesion to the roots of seven grasses and five cereals. Type 3-fimbriated Klebsiella strains adhered better than the other strains, and type 3 fimbriae appeared to be major adhesins for the Klebsiella strains. Although variations between plants were observed, no host specificity for bacterial adhesion was found.     

Diversity of bacteria in different life stages and their impact on the development and reproduction of Zeugodacus tau (Diptera: Tephritidae)

Fruit flies usually harbor diverse communities of bacteria in their digestive systems,which are known to play a significant role in their fitness.However,little information is available on Zeugodacus tau,a polyphagous pest worldwide.This study reports the first extensive analysis of bacterial communities in different life stages and their effect on the development and reproduction of laboratory-reared Z tan.Cultured bacteria were identified using the conventional method and all bacteria were identified by highthroughput technologies(16S ribosomal RNA gene sequencing of V3-V4 region).A total of six bacterial phyla were identified in larvae,pupae,and male and female adult flies,which were distributed into 14 classes,32 orders,58 families and 96 genera.Proteobacteria was the most represented phylum in all the stages except larvae.Enterobacter,Klebsiella,Providencia,and Pseudomonas were identified by conventional and next-generation sequencing analysis in both male and female adult flies,and Enterobacter was found to be the main genus.After being fed with antibiotics from the first instar larvae,bacterial diversity changed markedly in the adult stage.Untreated flies laid eggs and needed 20 days before oviposition while the treated flies showed ovary development inhibited and were not able to lay eggs,probably due to the alteration of the microbiota.These findings provide the cornerstone for unexplored research on bacterial function in Z tau,which will help to develop an environmentally friendly management technique for this kind of harmful insect.     

Insecticidal bacteria isolated from predatory larvae of the antlion species Myrmeleon bore (Neuroptera: Myrmeleontidae)

Various bacterial species were isolated from the crop (digestive organ) of the antlion species Myrmeleon bore and tested for their insecticidal activity against caterpillars by injection. Sixty-eight isolates from the antlion crop were grouped into twenty-four species based on homologies of 16S rRNA gene sequences and biochemical properties. Isolated Bacillus cereus, Bacillus sphaericus, Morganella morganii, Serratia marcescens and a Klebsiella species killed 80% or more cutworms when injected at a dose of 5x10(5)cells per insect. In addition, cutworms killed by these isolates resembled observations made of caterpillars attacked by antlions. A culture-independent analysis showed that the isolated bacterial species are likely to be frequently present in the antlion crop. These results suggest that insecticidal microorganisms associate with antlions, and may promote the death of prey.     

Evaluation of candidate probiotic strains for gilthead sea bream larvae (Sparus aurata) using an in vivo approach

AIMS: The aim of this study was to evaluate the effect of six bacterial strains on gilthead sea bream larvae (Sparus aurata). METHODS AND RESULTS: Six bacterial strains isolated from well-performing live food cultures were identified by sequencing fragments of their 16s rDNA genome to the genus level as Cytophaga sp., Roseobacter sp., Ruergeria sp., Paracoccus sp., Aeromonas sp. and Shewanella sp. Survival rates of gilthead sea bream larvae transferred to seawater added these bacterial strains at concentrations of 6 +/- 0.3 x 10(5) bacteria ml(-1) were similar to those of larvae transferred to sterilized seawater and showed an average of 86% at 9 days after hatching, whereas, survival rates of larvae transferred to filtered seawater were lower (P < 0.05), and showed an average of 39%, 9 days after hatching. CONCLUSION: Several bacterial strains isolated from well-performing live food cultures showed a positive effect for sea bream larvae when compared with filtered seawater. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach used in this study could be applied as an in vivo evaluation method of candidate probiotic strains used in the rearing of marine fish larvae.     

Phylogenetic characterization of bacteria in the gut of house flies (Musca domestica L.)

House flies (Musca domestica L.) are cosmopolitan, ubiquitous, synanthropic insects that serve as mechanical or biological vectors for various microorganisms. To fully assess the role of house flies in the epidemiology of human diseases, it is essential to understand the diversity of microbiota harbored by natural fly populations. This study aimed to identify the diversity of house fly gut bacteria by both culture-dependent and culture-independent approaches. A total of 102 bacterial strains were isolated from the gut of 65 house flies collected from various public places including a garden, public park, garbage/dump area, public toilet, hospital, restaurant/canteen, mutton shop/market, and house/human habitation. Molecular phylogenetic analyses placed these isolates into 22 different genera. The majority of bacteria identified were known potential pathogens of the genera Klebsiella, Aeromonas, Shigella, Morganella, Providencia, and Staphylococcus. Culture-independent methods involved the construction of a 16S rRNA gene clone library, and sequence analyses supported culture recovery results. However, additional bacterial taxa not determined via culture recovery were revealed using this methodology and included members of the classes Alphaproteobacteria, Deltaproteobacteria, and the phylum Bacteroidetes. Here, we show that the house fly gut is an environmental reservoir for a vast number of bacterial species, which may have impacts on vector potential and pathogen transmission.     

Exploration of host-pathogen interactions using Listeria monocytogenes and Drosophila melanogaster

The facultative intracellular bacterial pathogen Listeria monocytogenes is capable of replicating within a broad range of host cell types and host species. We report here the establishment of the fruit fly Drosophila melanogaster as a new model host for the exploration of L. monocytogenes pathogenesis and host response to infection. Listeria monocytogenes was capable of establishing lethal infections in adult fruit flies and larvae with extensive bacterial replication occurring before host death. Bacteria were found in the cytosol of insect phagocytic cells, and were capable of directing host cell actin polymerization. Bacterial gene products necessary for intracellular replication and cell-to-cell spread within mammalian cells were similarly found to be required within insect cells, and although previous work has suggested that L. monocytogenes virulence gene expression requires temperatures above 30 degrees C, bacteria within insect cells were found to express virulence determinants at 25 degrees C. Mutant strains of Drosophila that were compromised for innate immune responses demonstrated increased susceptibility to L. monocytogenes infection. These data indicate L. monocytogenes infection of fruit flies shares numerous features of mammalian infection, and thus that Drosophila has the potential to serve as a genetically tractable host system that will facilitate the analysis of host cellular responses to L. monocytogenes infection.     

Culture-independent characterization of the microbiota of the ant lion Myrmeleon mobilis (Neuroptera: Myrmeleontidae)

Ant lions are insect larvae that feed on the liquefied internal components of insect prey. Prey capture is assisted by the injection of toxins that are reportedly derived from both the insect and bacterial symbionts. These larvae display interesting gut physiology where the midgut is not connected to the hindgut, preventing elimination of solid waste until adulthood. The presence of a discontinuous gut and the potential involvement of bacteria in prey paralyzation suggest an interesting microbial role in ant lion biology; however, the ant lion microbiota has not been described in detail. We therefore performed culture-independent 16S rRNA gene sequence analysis of the bacteria associated with tissues of an ant lion, Myrmeleon mobilis. All 222 sequences were identified as Proteobacteria and could be subdivided into two main groups, the alpha-Proteobacteria with similarity to Wolbachia spp. (75 clones) and the gamma-Proteobacteria with similarity to the family Enterobacteriaceae (144 clones). The Enterobacteriaceae-like 16S rRNA gene sequences were most commonly isolated from gut tissue, and Wolbachia-like sequences were predominant in the head and body tissue. Fluorescence in situ hybridization analyses supported the localization of enterics to gut tissue and Wolbachia to nongut tissue. The diversity of sequences isolated from freshly caught, laboratory-fed, and laboratory-starved ant lions were qualitatively similar, although the libraries from each treatment were significantly different (P = 0.05). These results represent the first culture-independent analysis of the microbiota associated with a discontinuous insect gut and suggest that the ant lion microbial community is relatively simple, which may be a reflection of the diet and gut physiology of these insects.     

The tsetse fly Glossina fuscipes fuscipes (Diptera: Glossina) harbours a surprising diversity of bacteria other than symbionts

Three different bacterial species are regularly described from tsetse flies. However, no broad screens have been performed to investigate the existence of other bacteria in this medically and agriculturally important vector insect. Utilising both culture dependent and independent methods we show that Kenyan populations of Glossina fuscipes fuscipes harbour a surprising diversity of bacteria. Bacteria were isolated from 72% of flies with 23 different bacterial species identified. The Firmicutes phylum dominated with 16 species of which seven belong to the genus Bacillus. The tsetse fly primary symbiont, Wigglesworthia glossinidia, was identified by the culture independent pathway. However, neither the secondary symbiont Sodalis nor Wolbachia was detected with either of the methods used. Two other bacterial species were identified with the DNA based method, Bacillus subtilis and Serratia marcescens. Further studies are needed to determine how tsetse flies, which only ever feed on vertebrate blood, pick up bacteria and to investigate the possible impact of these bacteria on Glossina longevity and vector competence.     

染料脱色菌的分子分类和有关基因与其脱色特性的关系

从印染废水处理系统中分离到12株具有不同脱色特性的细菌,比较分析这12株细菌的质粒携带情况、分子分类地位和黄素还原酶基因(fre)的拷贝数与其脱色特性之间的关系。结果发现,所分离到的这12株菌分别归属于希瓦氏菌属、克雷伯氏菌属、假单胞菌属、气单胞菌属和苍白杆菌属。具有较广谱脱色能力的细菌在系统进化树上基本聚为一类,分别归属于希瓦氏菌属和气单胞菌属,其质粒携带率也较低。fre基因在不同菌属中的基因型存在很大的差异,仅仅采用一套引物无法对不同菌属中的fre基因进行有效的扩增。     

The role of the different fermentation media and infection processes on the infectivity of the bacterial symbionts to Spodoptera littoralis (biosduval) (Lipedoptera: Noctuidae)

Abstract

Bacterial symbionts are one of the most toxic bacteria to insect pests. They have been isolated from the intestine of the entomopathogenic nematodes. Ten strains of the entomopathogenic nematodes, that may be different, have been isolated out of different soil fauna having different crops from different Governorates in Egypt. The bacterial symbionts in these strains have been isolated and tested for production parameters using four different media. Experiments showed that both Loria Broth (LB), and Nutrient Broth (NB) gave good results in laboratory production of bacterial symbionts, concerning stability, cell size, and pigment production during culturing. Experiments using different techniques of introducing the bacterial symbionts to larvae of the cotton leafworm showed that the injection technique was the most effective among all the tested techniques. This is followed by oral and forced feeding which seemed to give equal results. On the other hand, toxicity experiments showed that the four bacterial isolates named, G-NRC-A₃, SH-NRC-A₅, SH-NRC-A₆, and N.Sinai-NRC-A₈, besides the bacterial symbiont of Steinernema abbasi, Flavimonas oryzihabitans, all give 100% mortalities to Spodoptera littoralis larvae after 24 h post-treatment at the higher dose (5×10⁴ cells/10 μl) but at the lower doses 5×10³ and 5×10² cells/10 μl of injection solution, a 100% mortality was reached after 72 h post-treatment.     

Activation or detoxification of mutagenic and carcinogenic compounds in transgenic Drosophila expressing human glutathione S-transferase.

Sensitivity of transgenic Drosophila melanogaster with expression of a human gene encoding the glutathione S-transferase alpha subunit (GSTA1-1) to 1,2:5,6-dibenzanthracene (DBA) and 1,2-dichloroethane (DCE) was investigated in the somatic mutation and recombination test (SMART). We performed the same assay in control transgenic flies expressing the bacterial lacZ gene. Three types of transgenic Drosophila strains carrying GSTA1-1 were used: two transgenic strains homozygous for the second chromosome with a single-copy transgene insertion and one strain with two transgene insertions. Larvae carrying the lacZ gene were significantly more sensitive to genotoxic effects of DBA than those carrying three copies of the GSTA1-1 gene. The larvae with lacZ expression showed significantly lower sensitivity to DCE compared with those expressing GSTA1-1. Finally, a pretreatment with buthionine-sulphoximine (BSO) in experiment with DCE significantly decreased the frequency of mutation events in larvae with three GSTA1-1 copies in comparison with others.     

Molecular cloning, genomic organization, and expression of a C-type (Manduca sexta-type) allatostatin preprohormone from Drosophila melanogaster

The insect allatostatins are a diverse group of neuropeptides that obtained their names by their inhibitory actions on the corpora allata (two endocrine glands near the insect brain), where they block the biosynthesis of juvenile hormone (a terpenoid important for development and reproduction). Chemically, the allatostatins can be subdivided into three different peptide groups: the large group of A-type (cockroach-type) allatostatins, which have the common C-terminal sequence Y/FXFGLamide; the B-type (cricket-type) allatostatins, which have the C-terminal sequence W(X(6))Wamide in common; and a single allatostatin that we now call C-type allatostatin that was first discovered in the moth Manduca sexta, and which has a nonamidated C terminus, and a structure unrelated to the A- and B-type allatostatins. We have previously cloned the preprohormones for the A- and B-type allatostatins from Drosophila melanogaster. Here we report on the cloning of a Drosophila C-type allatostatin preprohormone (DAP-C). DAP-C is 121 amino acid residues long and contains one copy of a peptide sequence that in its processed form has the sequence Y in position 4) from the Manduca sexta C-type allatostatin. The DAP-C gene has three introns and four exons and is located at position 32D2-3 on the left arm of the second chromosome. Northern blots show that the gene is strongly expressed in larvae and adult flies, but less in pupae and embryos. In situ hybridizations of larvae show that the gene is expressed in various neurons of the brain and abdominal ganglia and in endocrine cells of the midgut. This is the first publication on the structure of a C-type allatostatin from insects other than moths and the first report on the presence of all three types of allatostatins in a representative of the insect order Diptera (flies).     

The cutworm Peridroma saucia (Lepidoptera: Noctuidae) supports growth and transport of pBR322-bearing bacteria

Variegated cutworms were exposed to bean plants in microcosms sprayed with pBR322-carrying strains of Enterobacter cloacae, Klebsiella planticola, and Erwinia herbicola. The three bacterial species exhibited differential survival on leaves, in soil, and in guts and fecal pellets (frass) of the insects. High numbers of Enterobacter cloacae(pBR322) were detected in all samples, while the other species were unable to establish residence in the insect. To assess the impact of this colonization on site-to-site transport of microorganisms, larvae were fed plants that had been sprayed with the bacteria and then were transferred to uninoculated plants. Cutworms were efficient carriers of Enterobacter cloacae(pBR322), as indicated by its rapid appearance on uninoculated leaves and continued persistence in the insects for 3 days after transfer. Few Erwinia herbicola(pBR322) and K. planticola(pBR322) were obtained from larvae after transfer, although up to 10(3) CFU/g were detected in soil and on plants. Differences in bacterial survival and growth were confirmed by incubating frass overnight and observing the change in population numbers. The proportion of total samples showing at least a 25-fold increase during incubation was 68% for Enterobacter cloacae(pBR322), 39% for K. planticola(pBR322), and 0% for Erwinia herbicola(pBR322). Our results emphasize the role that cutworms and possibly other insects have in persistence and growth of microorganisms in the environment.     

The cutworm Peridroma saucia (Lepidoptera: Noctuidae) supports growth and transport of pBR322-bearing bacteria.

Variegated cutworms were exposed to bean plants in microcosms sprayed with pBR322-carrying strains of Enterobacter cloacae, Klebsiella planticola, and Erwinia herbicola. The three bacterial species exhibited differential survival on leaves, in soil, and in guts and fecal pellets (frass) of the insects. High numbers of Enterobacter cloacae(pBR322) were detected in all samples, while the other species were unable to establish residence in the insect. To assess the impact of this colonization on site-to-site transport of microorganisms, larvae were fed plants that had been sprayed with the bacteria and then were transferred to uninoculated plants. Cutworms were efficient carriers of Enterobacter cloacae(pBR322), as indicated by its rapid appearance on uninoculated leaves and continued persistence in the insects for 3 days after transfer. Few Erwinia herbicola(pBR322) and K. planticola(pBR322) were obtained from larvae after transfer, although up to 10(3) CFU/g were detected in soil and on plants. Differences in bacterial survival and growth were confirmed by incubating frass overnight and observing the change in population numbers. The proportion of total samples showing at least a 25-fold increase during incubation was 68% for Enterobacter cloacae(pBR322), 39% for K. planticola(pBR322), and 0% for Erwinia herbicola(pBR322). Our results emphasize the role that cutworms and possibly other insects have in persistence and growth of microorganisms in the environment.     

Microflora species and their volatile compounds affecting development of an alcohol dehydrogenase homozygous strain (Adh-I) of Bactrocera (Dacus) oleae (Diptera: Tephritidae)

Microflora species and volatiles emitted from artificial diets were examined from the larvae of three homozygous alcohol dehydrogenase (Adh) strains of the olive fruit fly, Bactrocera (Dacus) oleae (Gmelin), reared under identical conditions. Differences in volatile composition were detected when Adh-I homozygous larvae developed in a diet lacking the preservative p-hydroxybenzoic acid methyl ester (nipagin). Larval development of the Adh-I strain in the preservative-free diet was reduced by 50%, whereas pupal emergence was completely inhibited. The larval development and pupal emergence of Adh-F and Adh-S strains were not affected. Unique microorganisms with characteristic volatile profiles were isolated from the preservative-free diet of the Adh-I strain that were different from those, isolated from Adh-S, Adh-F, laboratory colony, and wild insect populations. Our results indicated that the variations in volatile composition of the artificial diets, and the inhibition of larval development and pupal emergence in Adh-I strain were related to changes in the microflora that developed in the diets of the Adh-I strain.