Recent Advanced Methods to Study Bacterial in Insects

Presented by Dr. V.S. Saravanan Ph.D. Asst. Professor Dept. of Microbiology Indira Gandhi College of Arts and Science Pondicherry

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 1 Topics Covered

• What are endosymbionts? • Culturable Vs Uncultivated bacterial endosymbionts • Structural community composition studies Vs Functional studies • Insect gut • Cultivation dependent approaches • Cultivation independent approaches – Community analysis, metagenome analysis and genome • Out come – Community profiles & Functional significance

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 2 What are endosymbionts?

• Term endosymbionts refers to microbes present intracellular in the cells or tissues of the animals. • Bacteriocytes - is a specialized adipocyte tissue found primarily in certain insects such as , tsetse flies, German cockroaches and weevils. • These cells contain endosymbiotic organisms such as and yeast, which provide essential amino acids and other chemicals to their host. • Bacteriocytes may aggregate into a specialized organ called the bacteriome. (Baumann, 2005)

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 3 Intracellular in Insects (Kikuchi, 2009) Symbiont Phylum / Class Host Host feeding nature Host association Host interaction Transmission mechanism

Intracellular symbionts

Buchnera aphidicola* γ - Aphids Phytophagous Obligate Mutualistic Transovarial Carsonella ruddii* γ -Proteobacteria Psyllids Phytophagous Obligate Mutualistic Transovarial Portiera aleyrodidarum* γ -Proteobacteria Whiteflies Phytophagous Obligate Mutualistic Transovarial Tremblaya princeps*  -Proteobacteria Mealy bugs Phytophagous Obligate Mutualistic Transovarial Baumannia cicadellinicola* γ -Proteobacteria Sharpshooters Phytophagous Obligate Mutualistic Transovarial Sulcia muelleri* Bacteroidetes Sharpshooters Phytophagous Obligate Mutualistic Transovarial Nardonella spp. * γ -Proteobacteria Weevils Phytophagous Obligate Mutualistic Transovarial Sitophilus symbiont (SOPE) * γ -Proteobacteria Weevils Phytophagous Obligate Mutualistic Transovarial Wigglesworthia glossinidia* γ -Proteobacteria Tsetse flies Phytophagous Obligate Mutualistic Milk-gland Blochmannia floridanus* γ -Proteobacteria Carpenter ants Omnivorous Obligate Mutualistic Transovarial Serratia symbiotica# γ -Proteobacteria Aphids Phytophagous Facultative Commensalistic Transovarial Regiella insecticola** γ -Proteobacteria Aphids Phytophagous Facultative Commensalistic Transovarial # γ -Proteobacteria Aphids Phytophagous Facultative Commensalistic Transovarial Fritschea spp.* Chlamydiae Whiteflies Phytophagous Facultative Commensalistic Transovarial Sodalis glossinidius# γ -Proteobacteria Tsetse flies Hematophagous Facultative Commensalistic Milk-gland

Arsenophonus arthropodicus# γ -Proteobacteria Louse flies Hematophagous Facultative Commensalistic Transovarial

Arsenophonus triatominarum** γ -Proteobacteria Assassin bug Hematophagous Facultative Commensalistic Transovarial Wolbachia spp.** -Proteobacteria Various Facultative Parasitic Transovarial Rickettsia spp. ** -Proteobacteria Various insects Various Facultative Parasitic Transovarial Spiroplasma spp.* Firmicutes Various insects Various Facultative Parasitic Transovarial Cardinium spp. ** Bacteroidetes Various insects Various Facultative Parasitic Transovarial * - Unculturable; ** - co-culturing with insect cell lines; # - culturable in axenic media 09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 4 Structural community composition Vs Functional Studies

Structural Community Composition

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 5 Structural community composition studies • Cultivable (Great Plate Count Anomaly – Staley and Knopka, 1985) • Total bacterial population [cultivable Vs uncultivated (uncultivable)]

Pederin A blistering agent found in the haemolymph of the beetle genus Paederus

Functional studies - Screening for functional genes like cellulase, lignin peroxidase 09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 6 Coxiella a Rickettsia, isolated from Ticks

Wolbachia melophagi (Nöller 1917) Wolbachia persica (Suitor and Weiss 1961) Wolbachia pipientis (Hertig 1936)

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 7 Hindgut is the region bearing the largest microbial populations In particular, the ileum (the region between the proximal pylorus and distal rectum) is a relatively favourable environment (Douglas, 2015)

 It lacks the digestive enzymes of the midgut  Desiccation stress of the distal hindgut, where water is actively resorbed from the lumen into insect tissues.  Ions and metabolites delivered to the hindgut in the filtrate from the malpighian tubules.

 Firmicutes are abundant in the termite Nasutitermes corniger, but the dominant representatives of this phylum vary with gut region (Lactobacillales in the crop, Lachnospiraceae in the midgut, and Ruminococcaceae in the distal hindgut)

 Unusually, the dipteran olive fly Bactrocera oleae has an esophageal evagination, known as the cephalic bulb (near crop), which houses a dense culture of a single bacterium, “Candidatus Erwinia dacicola (Capuzzo et al., 2005).

 Behavioral adaptations that further promote the dominant gut microbial taxa, including coprophagy, trophallaxis (transfer of gut fluids by anus-to-mouth or mouth-to-mouth feeding), and maternal smearing of gut microorganisms on the eggshell, which is subsequently consumed by the offspring 09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 8 Cultivation dependent

• Objective is towards study of bacterial population by cultivation - insect gut is dissected. - suspended in phosphate buffered saline - serially diluted - plated on suitable media, incubated 28 °C for 48 h, colonies analyzed E.g. Spiroplasma poulsonii (Drosophila) have been cultivated in cell-free media as axenic culture. - Barbour- Stoenner-Kelly H (BSK-H), this medium was supplemented with enriched nutrients as predicted from the genome of S. poulsonii, - further success of cultivation - optimizing external growth factors like pH, partial pressure in O2, fly extract supplementation and lipid supplementation (Massan et al., 2018).

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 9 Cultivation Dependent Community Analysis

. Sodalis glossidinus (Tsetse flies), . Arsenophonus arthropodicus (Louse flies), . Serratia symbiotica (Aphids), . Hamiltonella defensa (Aphids) and . Spiroplasma poulsonii (Drosophila) have been cultivated in cell-free media as axenic culture 09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 10 Engel & Moran, 2013 09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 11 Cultivation independent

• Objective - entire bacterial population (cultivable as well as uncultivable) without cultivation meant - individual gut or pooled guts from 10 or 30 larvae or adult insects are placed in 1.5 mL microfuge tubes containing 50 µ L PBS and maintained at 4°C until DNA extraction. • Density gradient methods were generally useful to enrich the microorganisms associated with plant tissues or soil particles, in particular Nycodenz density gradient method was successfully used to separate bacterial cells from soil particles (Curotois et al., 2001).

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 12 Contd.,

• In the cell recovery method of metagenomes collection, intact microorganism (gut content) - frequent homogenization and differential centrifugation or by gradient centrifugation in media such as percoll or sucrose • The bacterial cells - separated from the insect cells - through filtration (e.g.1m). • The isolated samples could be selectively maintained as enrichment by selective pressure like nutritional selective conditions or supplying limiting nutrient (Krishnan et al. 2014).

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 13 Contd.

• In direct lysis method of metagenomes collection, gentle enzymatic lysis of insect gut cells - intact microbial cell washing - lysing by thermal shocks, homogenization and bead beating. • High molecular weight DNA - metagenomic library construction – its free of macromolecules – hindering restriction digestion, PCR amplification and cloning. • Most of the gut metagenomic DNA extraction procedure has been adopted from soil DNA isolation methods with slight modifications.

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 14 Cultivation Independent Community Analysis

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 15 09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 16 Shotgun sequencing Approach comparing the use in Metagenome and Genomic Approaches

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 17 Krishnan et al., 2014 09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 18 Community composition

• Four bacterial phyla (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria) dominant. • Methanoarchaeota (methanogens) - non methanogenic Thermoplasmatales and Halobacteriales - hindgut of cockroaches (order Blattodea). • Pantoea spp. () - from both environmental samples (e.g., water, soil, plant material) and insects, including mosquitoes, thrips, bees and hemipterans (Valiente Moro et al., 2013)

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 19 Community composition (contd.)

• Members of Acetobacteraceae (Alphaproteobacteria) - fruits and fermented foods and beverages, - guts of insects feeding on sugar- rich diets; e.g., bees, fruit flies and mosquitoes (Crotti et al., 2010). • Insect-specific species of gut bacteria - e.g., Snodgrassella alvi (Betaproteobacteria) and Gilliamella apicola (Gammaproteobacteria) in honey bees (Apis mellifera)

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 20 Functional significance

• The psyllid Bactericerca cockerelli (Sulc) - symbiont to modulate plant defensive gene expression. • Maintenance of leaf green islands by symbiotic bacteria which is fundamental for leaf miners’ survival. • Sequencing of diamondback moth (Plutella xylostella L.) metagenome, together with the functional profile of its microbiota, revealed a major role of bacterial taxa in the adaptation to detoxify plant defense compounds

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 21 Functional significance (contd.,)

• A metatranscriptomic approach showed the causal agent of colony collapse disorder in honeybees, a correlation between the disease and Israeli acute paralysis virus was noticed. Viral population based decline of the invasive yellow crazy ant (Anoplolepis gracilipes Smith) was noticed in Australia (Cox-Foster et al. 2007; Cooling et al. 2017). • A metagenomics approach has been used to study the viral community of different species of mosquitoes, revealing a very diverse community of animal, plant, insect and bacterial viruses (Ng et al., 2007). All these studies could pay way for developing novel pest management programmes using viruses.

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 22 Functional significance (contd.,)

• The analysis of the gut microbiota of Hyles euphorbiae (L.) and Brithys crini , revealed a community dominated by Enterococcus, with a likely role in helping these insects to feed on toxic plants. • Insect-microorganism relationships could be manipulated to improve pest control, by decreasing pests’ fitness or by increasing the efficacy of pest management programs. One possible target are bacteria (e.g., Hamiltonella defensa, Regiella insecticola) protecting whiteflies and aphids from natural enemies (parasitoids) attack • DNA metabarcoding has been shown to be useful in the identification of botanical and entomological sources of honey, a valuable product subject to fraud (Prosser and Hebert, 2017)

09-Dec-19 ICAR-CAFT - Recent advances in Endosymbionts of Plants & Insects 23 References Baumann, P. (2005) Biology bacteriocyte-associated endosymbionts of plant sap-sucking insects. Annu. Rev. Microbiol. 59:155–189.

Capuzzo C, Firrao G, Mazzon L, Squartini A, Girolami V. (2005) Candidatus Erwinia dacicola’, a co-evolved symbiotic bacterium of the olive fly Bactrocera oleae (Gmelin) Int J Syst Evol Microbiol. 55:1641–1647

Cooling M., Gruber M.A.M., Hoffmann B.D., Sébastien A., Lester P.J. A metatranscriptomic survey of the invasive yellow crazy ant, Anoplolepis gracilipes, identifies several potential viral and bacterial pathogens and mutualists. Insectes Soc. 2017;64:197–207.

Courtois S, Frostegard A, Göransson P, Depret G, Jeannin P, Simonet P. Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation. Environ Microbiol 2001; 3: 431-439.

Cox-Foster D.L., Conlan S., Holmes E.C., Palacios G., Evans J.D., Moran N.A., Quan P.-L., Briese T., Hornig M., Geiser D.M. (2007) A metagenomic survey of microbes in honey bee colony collapse disorder. Science. 2007;318:283–287.

Crotti E, Rizzi A, Chouaia B, Ricci I, Favia G, (2010) Acetic acid bacteria, newly emerging symbionts of insects. Appl Environ Microbiol.76:6963–6970.

Douglas A.E (2015) Multiorganismal insects: Diversity and function of resident microorganisms. Annu. Rev. Entomol., 60: 17-34.

Kikuchi Y. (2009) Endosymbiotic bacteria in Insects: Their diversity and culturability. Microbes Environ. 24: 195-204.

Krishnan M., Bharathiraja C., Pandiarajan J., Arun Prasanna V., Rajendhran J., Gunasekaran P. (2014) Insect gut microbiome - An unexploited reserve for biotechnological. Application. Asian Pac. J. Trop. Biomed. 4: S16- 521.

Masson F., Copete S.C., Schűpfer F., Garcia-Araez G. and Lemaitre B. (2018) and In Vitro culture of the insect endosymbiont Spiroplasma poulsonii highlights bacterial genes involved in host symbiont interaction. mBio. 9: e00024-18.

Ng T.F.F., Willner D.L., Lim Y.W., Schmieder R., Chau B., Nilsson C., Anthony S., Ruan Y., Rohwer F., Breitbart M. (2011) Broad surveys of DNA viral diversity obtained through viral metagenomics of mosquitoes. PLoS ONE. 6:e20579.

Prosser S.W.J., Hebert P.D.N. Rapid identification of the botanical and entomological sources of honey using DNA metabarcoding. Food Chem. 2017;214:183–191.

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Staley JT and Konopka A. (1985) Measurements of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol. 39:321-346

Valiente Moro C, Tran FH, Raharimalala FN, Ravelonandro P, Mavingui P. (2013) Diversity of culturable bacteria including Pantoea in wild mosquito Aedes albopictus. BMC Microbiol. 13:70

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