The Importance of Environmentally Acquired Bacterial Symbionts for the Squash Bug (Anasa tristis), a Significant Agricultural Pest

Most insects maintain associations with microbes that shape their ecology and evolution. Such symbioses have important applied implications when the associated insects are pests or vectors of disease. The squash bug, Anasa tristis (Coreoidea: Coreidae), is a significant pest of human agriculture in its own right and also causes damage to crops due to its capacity to transmit a bacterial plant pathogen. Here, we demonstrate that complete understanding of these insects requires consideration of their association with bacterial symbionts in the family Burkholderiaceae. Isolation and sequencing of bacteria housed in the insects’ midgut crypts indicates that these bacteria are consistent and dominant members of the crypt-associated bacterial communities. These symbionts are closely related to Caballeronia spp. associated with other true bugs in the superfamilies Lygaeoidea and Coreoidea. Fitness assays with representative Burkholderiaceae strains indicate that the association can significantly increase survival and decrease development time, though strains do vary in the benefits that they confer to their hosts, with Caballeronia spp. providing the greatest benefit. Experiments designed to assess transmission mode indicate that, unlike many other beneficial insect symbionts, the bacteria are not acquired from parents before or after hatching but are instead acquired from the environment after molting to a later developmental stage. The bacteria do, however, have the capacity to escape adults to be transmitted to later generations, leaving the possibility for a combination of indirect vertical and horizontal transmission.

The importance of environmentally-acquired bacterial symbionts for the squash bug (Anasa tristis), a significant agricultural pest

Most insects maintain associations with microbes that shape their ecology and evolution. Such symbioses have important applied implications when the associated insects are pests or vectors of disease. The squash bug, Anasa tristis (Coreoidea: Coreidae), is a significant pest of human agriculture in its own right and also causes damage to crops due to its capacity to transmit a bacterial plant pathogen. Here, we demonstrate that complete understanding of these insects requires consideration of their association with bacterial symbionts in the family Burkholderiaceae. Isolation and sequencing of bacteria housed in midgut crypts in these insects indicates that these bacteria are consistent and dominant members of the crypt-associated bacterial communities. These symbionts are closely related to Caballeronia spp. associated other true bugs in the superfamiles Lygaeoidea and Coreoidea. Fitness assays with representative Burkholderiaceae strains indicate that the association can significantly increase survival and decrease development time, though strains do vary in the benefits that they confer to their hosts, with Caballeronia spp. providing the greatest benefit. Experiments designed to assess transmission mode indicate that unlike many other beneficial insect symbionts, the bacteria are not acquired from parents before or after hatching but are instead acquired from the environment after molting to a later development stage. The bacteria do, however, have the capacity to escape adults to be transmitted to later generations, leaving the possibility for a combination of indirect vertical and horizontal transmission.

Insecticide resistance governed by gut symbiosis in a rice pest, Cletus punctiger, under laboratory conditions

Resistance to toxins in insects is generally thought of as their own genetic trait, but recent studies have revealed that gut microorganisms could mediate resistance by detoxifying phytotoxins and man-made insecticides. By laboratory experiments, we here discovered a striking example of gut symbiont-mediated insecticide resistance in a serious rice pest, Cletus punctiger . The rice bug horizontally acquired fenitrothion-degrading Burkholderia through oral infection and housed it in midgut crypts. Fenitrothion-degradation test revealed that the gut-colonizing Burkholderia retains a high degrading activity of the organophosphate compound in the insect gut. This gut symbiosis remarkably increased resistance against fenitrothion treatment in the host rice bug. Considering that many stinkbug pests are associated with soil-derived Burkholderia , our finding strongly supports that a number of stinkbug species could gain resistance against insecticide simply by acquiring insecticide-degrading gut bacteria.

Complete Genome Sequence of Burkholderia sp. Strain THE68, a Bacterial Symbiont Isolated from Midgut Crypts of the Seed Bug Togo hemipterus

Burkholderia sp. strain THE68 is a bacterial symbiont isolated from the midgut crypts of a phytophagous stink bug, Togo hemipterus. Here, we report the complete 7.98-Mb genome of this symbiont, which consists of six circular replicons containing 7,238 protein coding genes.

Host–symbiont specificity determined by microbe–microbe competition in an insect gut

Despite the omnipresence of specific host–symbiont associations with acquisition of the microbial symbiont from the environment, little is known about how the specificity of the interaction evolved and is maintained. The bean bug Riptortus pedestris acquires a specific bacterial symbiont of the genus Burkholderia from environmental soil and harbors it in midgut crypts. The genus Burkholderia consists of over 100 species, showing ecologically diverse lifestyles, and including serious human pathogens, plant pathogens, and nodule-forming plant mutualists, as well as insect mutualists. Through infection tests of 34 Burkholderia species and 18 taxonomically diverse bacterial species, we demonstrate here that nonsymbiotic Burkholderia and even its outgroup Pandoraea could stably colonize the gut symbiotic organ and provide beneficial effects to the bean bug when inoculated on aposymbiotic hosts. However, coinoculation revealed that the native symbiont always outcompeted the nonnative bacteria inside the gut symbiotic organ, explaining the predominance of the native Burkholderia symbiont in natural bean bug populations. Hence, the abilities for colonization and cooperation, usually thought of as specific traits of mutualists, are not unique to the native Burkholderia symbiont but, to the contrary, competitiveness inside the gut is a derived trait of the native symbiont lineage only and was thus critical in the evolution of the insect gut symbiont.

Culturing and Characterization of Gut Symbiont Burkholderia spp. from the Southern Chinch Bug, Blissus insularis (Hemiptera: Blissidae)

ABSTRACTThe phloem-feeding Southern chinch bug,Blissus insularis, harbors a high density of the exocellular bacterial symbiontBurkholderiain the lumen of specialized midgut crypts. Here we developed an organ culture method that initially involved incubating theB. insulariscrypts in osmotically balanced insect cell culture medium. This approach enabled the crypt-inhabitingBurkholderiaspp. to make a transition to anin vitroenvironment and to be subsequently cultured in standard bacteriological media. Examinations using ribotyping and BOX-PCR fingerprinting techniques demonstrated that mostin vitro-produced bacterial cultures were identical to their crypt-inhabitingBurkholderiacounterparts. Genomic and physiological analyses of gut-symbioticBurkholderiaspp. that were isolated individually from two separateB. insularislaboratory colonies revealed that the majority of individual insects harbored a singleBurkholderiaribotype in their midgut crypts, resulting in a diverseBurkholderiacommunity within each colony. The diversity was also exhibited by the phenotypic and genotypic characteristics of theseBurkholderiacultures. Access to cultures of crypt-inhabiting bacteria provides an opportunity to investigate the interaction between symbioticBurkholderiaspp. and theB. insularishost. Furthermore, the culturing method provides an alternative strategy for establishingin vitrocultures of other fastidious insect-associated bacterial symbionts.IMPORTANCEAn organ culture method was developed to establishin vitrocultures of a fastidiousBurkholderiasymbiont associated with the midgut crypts of the Southern chinch bug,Blissus insularis. The identities of the resulting cultures were confirmed using the genomic and physiological features ofBurkholderiacultures isolated fromB. insulariscrypts, showing that host insects maintained the diversity ofBurkholderiaspp. over multiple generations. The availability of characterized gut-symbioticBurkholderiacultures provides a resource for genetic manipulation of these bacteria and for examination of the mechanisms underlying insect-bacterium symbiosis.

Purine Biosynthesis, Biofilm Formation, and Persistence of an Insect-Microbe Gut Symbiosis

ABSTRACTTheRiptortus-Burkholderiasymbiotic system is an experimental model system for studying the molecular mechanisms of an insect-microbe gut symbiosis. When the symbiotic midgut ofRiptortus pedestriswas investigated by light and transmission electron microscopy, the lumens of the midgut crypts that harbor colonizingBurkholderiasymbionts were occupied by an extracellular matrix consisting of polysaccharides. This observation prompted us to search for symbiont genes involved in the induction of biofilm formation and to examine whether the biofilms are necessary for the symbiont to establish a successful symbiotic association with the host. To answer these questions, we focused onpurNandpurT, which independently catalyze the same step of bacterial purine biosynthesis. When we disruptedpurNandpurTin theBurkholderiasymbiont, the ΔpurNand ΔpurTmutants grew normally, and only the ΔpurTmutant failed to form biofilms. Notably, the ΔpurTmutant exhibited a significantly lower level of cyclic-di-GMP (c-di-GMP) than the wild type and the ΔpurNmutant, suggesting involvement of the secondary messenger c-di-GMP in the defect of biofilm formation in the ΔpurTmutant, which might operate via impaired purine biosynthesis. The host insects infected with the ΔpurTmutant exhibited a lower infection density, slower growth, and lighter body weight than the host insects infected with the wild type and the ΔpurNmutant. These results show that the function ofpurTof the gut symbiont is important for the persistence of the insect gut symbiont, suggesting the intricate biological relevance of purine biosynthesis, biofilm formation, and symbiosis.