Symbioses between insects and microbes are ubiquitous, but vary greatly in terms of function, transmission mechanism, and location in the insect. Lepidoptera (butterflies and moths) are one of the largest and most economically important insect orders; yet, in many cases, the ecology and functions of their gut microbiomes are unresolved. We used high-throughput sequencing to determine factors that influence gut microbiomes of field-collected fall armyworm ( Spodoptera frugiperda ) and corn earworm ( Helicoverpa zea ). Fall armyworm midgut bacterial communities differed from those of corn earworm collected from the same host plant species at the same site. However, corn earworm bacterial communities differed between collection sites. Subsequent experiments using fall armyworm evaluating the influence of egg source and diet indicated that that host plant had a greater impact on gut communities. We also observed differences between regurgitant (foregut) and midgut bacterial communities of the same insect host, suggesting differential colonization. Our findings indicate that host plant is a major driver shaping gut microbiota, but differences in insect physiology, gut region, and local factors can also contribute to variation in microbiomes. Additional studies are needed to assess the mechanisms that affect variation in insect microbiomes, as well as the ecological implications of this variability in caterpillars.
Dinucleoside polyphosphates act as agonists on purinergic P2Y receptors to mediate a variety of cellular processes. Symmetrical, naturally occurring purine dinucleotides are found in most living cells and their actions are generally known. Unsymmetrical purine dinucleotides and all pyrimidine containing dinucleotides, however, are not as common and therefore their actions are not well understood. To carry out a thorough examination of the activities and specificities of these dinucleotides, a robust method of synthesis was developed to allow manipulation of either nucleoside of the dinucleotide as well as the phosphate chain lengths. Adenosine containing dinucleotides exhibit some level of activity on P2Y 1 while uridine containing dinucleotides have some level of agonist response on P2Y 2 and P2Y 6 . The length of the linking phosphate chain determines a different specificity; diphosphates are most accurately mimicked by dinucleoside triphosphates and triphosphates most resemble dinucleoside tetraphosphates. The pharmacological activities and relative metabolic stabilities of these dinucleotides are reported with their potential therapeutic applications being discussed.
Mechanical damage caused by insect feeding along with components present in insect saliva and oral secretions are known to induce jasmonic acid-mediated defense responses in plants. This study investigated the effects of bacteria from oral secretions of the fall armyworm Spodoptera frugiperda on herbivore-induced defenses in tomato and maize plants. Using culture-dependent methods, we identified seven different bacterial isolates belonging to the family Enterobacteriacea from the oral secretions of field-collected caterpillars. Two isolates, Pantoea ananatis and Enterobacteriaceae-1, downregulated the activity of the plant defensive proteins polyphenol oxidase and trypsin proteinase inhibitors (trypsin PI) but upregulated peroxidase (POX) activity in tomato. A Raoultella sp. and a Klebsiella sp. downregulated POX but upregulated trypsin PI in this plant species. Conversely, all of these bacterial isolates upregulated the expression of the herbivore-induced maize proteinase inhibitor (mpi) gene in maize. Plant treatment with P. ananatis and Enterobacteriaceae-1 enhanced caterpillar growth on tomato but diminished their growth on maize plants. Our results highlight the importance of herbivore-associated microbes and their ability to mediate insect plant interactions differently in host plants fed on by the same herbivore.
A Crystallogral structure is described for the Mg2+-BeF3--bound receiver domain of Sinorhizobium meliloti DctD bearing amino acid substitution E121K. Differences between the apo- and ligand-bound active sites are similar to those reported for other receiver domains. However, the off and on states of the DctD receiver domain are characterized by dramatically different dimeric structures, which supports the following hypothesis of signal transduction. In the off state, the receiver domain and coiled-coil linker form a dimer that inhibits oligomerization of the AAA+ ATPase domain. In this conformation, the receiver domain cannot be phosphorylated or bind Mg2+ and BeF3-. Instead, these modifications stabilize an alternative dimeric conformation that repositions the subunits by approximately 20 A, thus replacing the a4-b5-a5 interface with an a4-b5 interface. Reoriented receiver domains permit the ATPase domain to oligomerize and stimulate open complex formation by the s54 form of RNA polymerase. NtrC, which shares 38% sequence identity with DctD, works differently. Its activated receiver domain must facilitate oligomerization of its ATPase domain. Significant differences exist in the signaling surfaces of the DctD and NtrC receiver domains that may help explain how triggering the common two-component switch can variously regulate assembly of a AAA+ ATPase domain.
In order to investigate the involvement of amino acids in the catalytic mechanism of the soluble epoxide hydrolase, different mutants of the murine enzyme were produced using the baculovirus expression system. Our results are consistent with the involvement of Asp-333 and His-523 in a catalytic mechanism similar to that of other alpha/beta hydrolase fold enzymes. Mutation of His-263 to asparagine led to the loss of approximately half the specific activity compared to wild-type enzyme. When His-332 was replaced by asparagine, 96.7% of the specific activity was lost and mutation of the conserved His-523 to glutamine led to a more dramatic loss of 99.9% of the specific activity. No activity was detectable after the replacement of Asp-333 by serine. However, more than 20% of the wild-type activity was retained in an Asp-333-->Asn mutant produced in Spodoptera frugiperda cells. We purified, by affinity chromatography, the wild-type and the Asp-333-->Asn mutant enzymes produced in Trichoplusia ni cells. We labeled these enzymes by incubating them with the epoxide containing radiolabeled substrate juvenile hormone III (JH III). The purified Asp-333-->Asn mutant bound 6% of the substrate compared to the wild-type soluble epoxide hydrolase. The mutant also showed 8% of the specific activity of the wild-type. Preincubation of the purified Asp-333-->Asn mutant at 37 degrees C (pH 8), however, led to a complete recovery of activity and to a change of isoelectric point (pI), both of which are consistent with hydrolysis of Asn-333 to aspartic acid. This intramolecular hydrolysis of asparagine to aspartic acid may explain the activity observed in this mutant. Wild-type enzyme that had been radiolabeled with the substrate was digested with trypsin. Using reverse phase-high pressure liquid chromatography, we isolated four radiolabeled peptides of similar polarity. These peptides were not radiolabeled if the enzyme was preincubated with a selective competitive inhibitor of soluble epoxide hydrolase 4-fluorochalcone oxide. This strongly suggested that these peptides contained a catalytic amino acid. Each peptide was characterized with N-terminal amino acid sequencing and electrospray mass spectrometry. All four radiolabeled peptides contained overlapping sequences. The only aspartic acid present in all four peptides and conserved in all epoxide hydrolases was Asp-333. These peptides resulted from cleavage at different trypsin sites and the mass of each was consistent with the covalent linkage of Asp-333 to the substrate.
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