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Persistence of the ground beetle (Coleoptera: Carabidae) microbiome to diet manipulation

19 Oct 2020-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: It is found that the ground beetle microbiome is consistent across different host food sources, and the observed similarity of the pygidial gland secretory cell microbiome across hosts suggests the possibility that it may be a conserved community, possibly due to functional interactions related to defensive chemistry.
Abstract: Host-associated microbiomes can play important roles in the ecology and evolution of their insect hosts, but bacterial diversity in many insect groups remains poorly understood. Here we examine the relationship between host environment, host traits, and microbial diversity in three species in the ground beetle family (Coleoptera: Carabidae), a group of roughly 40,000 species that synthesize a wide diversity of defensive compounds. This study found that the ground beetle microbiome is consistent across different host food sources. We used 16S amplicon sequencing to profile three species that are phylogenetically distantly related, trophically distinct, and whose defensive chemical secretions differ: Anisodactylus similis LeConte, 1851, Pterostichus serripes (LeConte, 1875), and Brachinus elongatulus Chaudoir, 1876. Wild-caught beetles were compared to individuals maintained in the lab for two weeks on carnivorous, herbivorous, or starvation diets. Soil environment but not diet had a significant effect on bacterial diversity and composition. The three carabid species have patterns of microbial diversity similar to those previously found in other insect hosts. Metagenomic samples from two highly active tissue types — guts, and pygidial gland secretory cells (which produce defensive compounds) — were processed and sequenced separately from those of the remaining body. The observed similarity of the pygidial gland secretory cell microbiome across hosts suggests the possibility that it may be a conserved community, possibly due to functional interactions related to defensive chemistry. These results provide a baseline for future studies of the role of microbes in the diversification of defensive chemical biosynthesis in carabids.

Summary (2 min read)

Introduction

  • Insects are by far the most diverse group of animals [1, 2], and it is becoming clear that the success of several major insect groups is due in part to their resident microbiomes [2, 3].
  • Microbiomes remain understudied in many major groups of insects, including Carabidae ground beetles.
  • If bacteria have contributed to the diversification of carabid beetle phenotypes, this connec- tion may be reflected in patterns of carabid microbiome composition and diversity.
  • Termites regulate unique microbiomes in each of several gut pouches [11], and many insect species maintain useful symbionts in specialized cells called bacteriocytes [28].

Beetle husbandry and dissection

  • Twelve individuals each of Anisodactylus similis LeConte, 1851, Pterostichus serripes (LeConte, 1875), and Brachinus elongatulus Chaudoir, 1876 were collected (total 36 specimens).
  • Banana-fed (Trader Joe’s, Dole Banana Ecuador) and mealworm-fed (Timberline, Vita-bugs Mini Mealworms 500 count) beetles were fed on the first day, and subsequently fed and watered every three days using heat-sterilized forceps and autoclaved water.
  • DNA was eluted by adding 50μL TB solution (10mM Tris) directly onto the beads and incubating for 5 minutes, then returning samples to the magnetic rack to pellet the SPRI beads and retrieve the DNA-containing supernatant.
  • The pooled library was purified (Qiagen Qiaquick PCR Purification Kit) and sent for Illumina MiSeq sequencing at the U.C. Berkeley Genomics Sequencing Laboratory.

Analysis

  • Amplicon reads for the V4 region of 16S were de-multiplexed with deML [35] and processed using DADA2 [36], including quality filtering with maxEE = 2.
  • Reads were de-replicated into unique 16S amplicon sequence variants (ASVs, also referred to as phylotypes) using a read error model parameterized from the data.
  • Sequence tables and taxonomic assignments were imported into R version 3.5 [38] for downstream analysis and combined into a single phyloseq [39] object for convenience.
  • Alpha diversity measures were calculated using the packages phyloseq [39] and picante [46].
  • Bray-Curtis distances were calculated both for aggregate community data and for the original dataset.

Alpha diversity

  • Phylogenetic diversity (PD), evenness, phylotype richness, and Shannon index were calculated for each sample.
  • PD of aggregate communities was not associated significantly with diet treatment.
  • Evidence of an effect of host species on microbial community diversity was rela- tively weak, and varied by tissue.
  • Secretory cells had a relatively consistent alpha diversity level across species, only varying significantly by the measure of evenness (p = 0.030).
  • Of the phylotypes present in wild-caught beetles, 78% were present in at least one other diet condition.

Community composition

  • The most abundant phyla across all samples were Proteobacteria (mean abundance 48.7%), Bacteroidetes (mean abundance 17.8%), Tenericutes, and Firmicutes.
  • Differences by host species arose more clearly at the level of bacterial genera, so community composition of each beetle species was plotted at this level (Fig 4).
  • Differential abundance analysis of secretory cells versus all other tissues revealed that four phylotypes associated with two families were differentially abundant (p< 0.002).
  • Compared to other tissues, gut microbiomes were more dominated by the ten most abundant bacterial genera; these ten genera composed over 50% of microbial abundance in all host species’ guts, and over 60% of abundance in B. elongatulus guts (Fig 4B).

Discussion

  • The present study assessed the extent to which the microbiomes of three carabid beetle species are influenced by short-term community turnover driven by diet.
  • The finding that carabid microbiomes are resilient to short-term dietary shifts does not help pinpoint the factors that are most important in microbial community assembly in ground beetles.
  • Previous research has found that factors such as environmental filtering [3] and routes of microbe dispersal [22] can shape microbiome composition, and the especially strong association with tissue type could be related to these factors.

Author Contributions

  • Kipling Will, Aman Gill, also known as Conceptualization.
  • Sean Perez, Melanie Gee, Bethany Xu, Shreeya Garg, also known as Investigation.
  • (C) Mean abundance of these bacterial genera in P. serripes, grouped by diet treatment.
  • Photographs of beetles depict typical host morphology.

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RESEARCH ARTICLE
Persistence of the ground beetle (Coleoptera:
Carabidae) microbiome to diet manipulation
Anita Silver
ID
1
, Sean Perez
ID
1
, Melanie Gee
1
, Bethany Xu
1
, Shreeya Garg
1
, Kipling Will
1,2
*,
Aman Gill
1
1 Department of Environmental Science, Policy and Management, University of California Berkeley,
Berkeley, California, United States of America, 2 Essig Museum of Entomology, University of California
Berkeley, Berkeley, California, United States of America
* kipwill@berkeley.edu
Abstract
Host-associated microbiomes can play important roles in the ecology and evolution of their
insect hosts, but bacterial diversity in many insect groups remains poorly understood. Here
we examine the relationship between host environment, host traits, and microbial diversity
in three species in the ground beetle family (Coleoptera: Carabidae), a group of roughly
40,000 species that synthesize a wide diversity of defensive compounds. This study used
16S amplicon sequencing to profile three species that are phylogenetically distantly related,
trophically distinct, and whose defensive chemical secretions differ: Anisodactylus similis
LeConte, 1851, Pterostichus serripes (LeConte, 1875), and Brachinus elongatulus Chau-
doir, 1876. Wild-caught beetles were compared to individuals maintained in the lab for two
weeks on carnivorous, herbivorous, or starvation diets (n = 3 beetles for each species-diet
combination). Metagenomic samples from two highly active tissue types—guts, and pygidial
gland secretory cells (which produce defensive compounds)—were processed and
sequenced separately from those of the remaining body. Bacterial composition and diversity
of these ground beetles were largely resilient to controlled changes to host diet. Different tis-
sues within the same beetle harbor unique microbial communities, and secretory cells in
particular were remarkably similar across species. We also found that these three carabid
species have patterns of microbial diversity similar to those previously found in carabid bee-
tles. These results provide a baseline for future studies of the role of microbes in the diversi-
fication of carabids.
Introduction
Insects are by far the most diverse group of animals [1, 2], and it is becoming clear that the suc-
cess of several major insect groups is due in part to their resident microbiomes [2, 3]. However,
microbiomes remain understudied in many major groups of insects, including Carabidae
ground beetles. Carabidae consists of around 40,000 described species, making it one of the
most species-rich animal families on earth [4]. Moreover, the variety of defensive chemicals
produced in the carabid pygidial gland system is an impressive example of evolutionary
PLOS ONE
PLOS ONE | https://doi.org/10.1371/journal.pone.0241529 March 19, 2021 1 / 16
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OPEN ACCESS
Citation: Silver A, Perez S, Gee M, Xu B, Garg S,
Will K, et al. (2021) Persistence of the ground
beetle (Coleoptera: Carabidae) microbiome to diet
manipulation. PLoS ONE 16(3): e0241529. https://
doi.org/10.1371/journal.pone.0241529
Editor: Omri Finkel, University of North Carolina at
Chapel Hill, UNITED STATES
Received: October 14, 2020
Accepted: March 3, 2021
Published: March 19, 2021
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0241529
Copyright: © 2021 Silver et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All data files are
available via the NCBI Sequence Read Archive
(BioProject PRJNA703093).
Funding: KW. NSF DEB #1556957. National
Science Foundation Division of Environmental

diversification [5]. Secretory cells of the pygidial gland system produce such diverse classes of
molecules as carboxylic acids, formic acid, quinones, hydrocarbons, and aromatics; chemical
diversity exists even within some genera [5]. Whether microbes play a functional role in cara-
bid chemical diversity has not yet been studied.
Interactions between insects and their associated microbiomes can contribute to insect
diversification [3]. Microbiomes can benefit host insects in many ways, such as producing vita-
min B [6], regulating host metabolism in response to stress [7], and contributing to host devel-
opment [8]. Notable examples of microbial symbionts supporting nutrient acquisition in
insects include Buchnera bacteria producing essential amino acids allowing aphids to live on a
nutrient-poor diet [9] and highly diverse termite gut microbes digesting cellulose for their
wood-feeding hosts [10, 11]. Unlike aphids and termites, carabids tend to be dietary general-
ists, but microbial species are also known to contribute to other host phenotypes, including
nutrient acquisition and detoxification. In ants [12], Harpalus pensylvanicus (Degeer, 1774)
(Carabidae) [13] and Cephaloleia (Coleoptera: Chrysomelidae) [14], microbial symbionts
assist their hosts in metabolizing different food sources. It is known that bacterial symbionts
enable several beetle species to thrive in chemically hostile environments. For example, the
mountain pine beetle Dendroctonus ponderosae (Hopkins, 1902) (Coleoptera: Curculionidae)
can inhabit pine trees because its microbes break down defensive terpenes produced by the
trees [15]. The microbiomes of Nicrophorus vespilloides Herbst, 1783 (Coleoptera: Silphidae)
and other carrion beetles protect their hosts from toxins and speed up host digestion, making
it easier for these beetles to feed on decaying carcasses [16, 17]. Insects are well known to bene-
fit from defensive and protective symbioses. Lagria villosa (F.) (Coleoptera: Tenebrionidae)
beetles live in symbiosis with Burkholderia gladioli that protect their host’s eggs from patho-
gens by producing the antifungal compound lagriamide [18]. Paederus (Coleoptera: Staphyli-
nidae) beetles are well known for producing toxic hemolymph that causes severe dermatitis;
the toxin, pederin, is produced by a Pseudomonas-like symbiont [19]. The Asian citrus psyllid
Diaphorina citri (Kuwafyama, 1908) (Hemiptera: Liviidae), an invasive pest in the U.S. that
causes citrus disease, harbors endosymbiotic Candidatus Profftella armatura (Betaproteobac-
teria) that produce diaphorin, a toxin similar to pederin [20].
In carabid beetles, pygidial gland secretory cells perform an important metabolic function
by synthesizing defensive chemicals for secretion. To our knowledge, no previous studies have
compared the microbial communities of secretory cells from different carabid species. Given
the multitude of established insect-bacterial associations as well as the diversity of ground bee-
tle defensive chemistry, it is worth investigating the microbial diversity of secretory cell tissues
as a first step in exploring potential functional links between secretory cell microbes and cara-
bid defensive chemical biosynthesis.
If bacteria have contributed to the diversification of carabid beetle phenotypes, this connec-
tion may be reflected in patterns of carabid microbiome composition and diversity. Insect
microbiome composition can be explained by several factors, such as host phylogeny [2, 21
23], dietary guild [23] or sampling locality [24]. Although many insects have persistent host-
associated communities, some do not, highlighting the potential for fluctuations in microbial
diversity; for example, some lepidoptera caterpillar microbiomes consist entirely of microbes
ingested with leaves, with constant turnover based on short-term diet [25]. If the carabid
microbiome is characterized by rapid, near-complete compositional turnover similar to that of
the caterpillar microbiome, then it would be sensitive to dietary shifts [12, 26] or other changes
to the local environment [11], and not obviously correlate with factors such as host phylogeny,
chemistry, or tissue type. The composition of persistent host-associated microbiomes can also
be influenced by changes to host diet, as has previously been found in some Coleoptera and
PLOS ONE
Host diet and microbial diversity in ground beetles
PLOS ONE | https://doi.org/10.1371/journal.pone.0241529 March 19, 2021 2 / 16
Biology. https://www.nsf.gov/bio/deb/about.jsp The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
Competing interests: The authors have declared
that no competing interests exist.

Lepidoptera species [14, 27], but would not exhibit near-complete compositional turnover as a
result of these short-term perturbations.
As insects have an open circulatory system that allows hemolymph to flow throughout the
body, microbial communities are found in many insect tissues [28]; but as in other animals,
insects often have distinct microbial communities in different tissues [10, 12, 28]. Some of this
diversity may relate to the variety of conditions found within insect anatomy, including aero-
bic and anaerobic regions and extreme pH gradients [11, 28]. Tissue-specific diversity could
also be explained by a co-evolutionary relationship between hosts and symbiotic microbiota,
in that hosts can harbor functionally useful bacteria in specialized tissues. For example, ter-
mites regulate unique microbiomes in each of several gut pouches [11], and many insect spe-
cies maintain useful symbionts in specialized cells called bacteriocytes [28]. The present study
focuses on the pygidial gland secretory cells (hereafter simply "secretory cells") and the gut. We
focus on the microbial communities of these tissues because they are responsible for defensive
chemical synthesis and digestion of food respectively—metabolic processes known to involve
bacterial symbionts in other insect taxa.
In this study, we used 16S metagenomic amplicon sequencing to quantify the bacterial diver-
sity hosted by three carabid species under several dietary treatments. Each host species produces
distinct primary defensive compounds: Anisodactylus similis produces formic acid, Pterostichus
serripes carboxylic acids, and Brachinus elongatulus quinones [29] [Will & Attygalle, unpublished
data]. Anisodactylus similus has a distinct natural feeding preference from the other two species,
so together these three species represent two different trophic types. Brachinus elongatulus and
P. serripes are naturally generalist predator-scavengers, preferring animal matter but observed in
nature and in the lab to eat a wide variety of sugar and protein rich plant and animal material; in
contrast, A. similis is typically observed feeding on fallen fruits, seeds, and pollen [30] [Will
unpbl.]. In addition to sequencing wild-caught beetles preserved at the time of collection, we
also subjected live beetles of each species to three controlled dietary treatments.
This preliminary study was intended to reveal how short-term dietary shifts in beetle hosts
affect bacterial diversity and composition, providing an initial step in broader efforts to charac-
terize carabid microbial diversity in relation to host diversification. If carabids lack established
microbiomes altogether, as seen in caterpillars [25], then diet treatment alone might explain a
significant amount of the variation across microbial communities. In that case, relative to con-
ditions observed in the field, same-host communities under different diet treatments might
diverge, and different-host communities under the same diet treatment might converge. On
the other hand, if carabids have resilient microbial communities—structured by environmen-
tal exposure at earlier life stages, vertical transmission, and/or symbiosis—we would expect
those communities to remain largely intact during controlled changes to host diet. Further, in
that case we expect that community diversity and composition would correlate with host char-
acteristics such as species, tissue type, or defensive chemistry. To better distinguish these possi-
ble associations, our study quantifies the effect of dietary shifts on several host species and
tissues separately. Notably, to our knowledge this study is the first to compare microbial com-
munities of carabid pygidial gland secretory cells from multiple species. This work provides a
baseline for future studies to investigate the connection between host-associated microbes and
carabid beetle phylogenetic and chemical diversity.
Methods
Beetle husbandry and dissection
Twelve individuals each of Anisodactylus similis LeConte, 1851, Pterostichus serripes (LeConte,
1875), and Brachinus elongatulus Chaudoir, 1876 were collected (total 36 specimens).
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Pterostichus serripes and A. similus were collected from U.C. Berkeley’s Whitaker’s Forest,
Tulare County, CA (36.7022˚, -118.933˚). Brachinus elongatulus were collected from national
forest land in Madera Canyon, Santa Cruz County, AZ (31.72˚, -110.88˚). The number of rep-
licate beetles used for this study was constrained by the practical difficulties of finding and
catching sufficient numbers of live specimens from multiple species. For each species, three
wild-caught specimens were preserved in 95% ethanol immediately upon collection, and the
remaining beetles were transported live to laboratory facilities on the U.C. Berkeley campus.
For each species, in addition to wild-caught specimens, three diet treatments (banana, meal-
worm, and starvation) were tested in triplicate. Diet-treated beetles were kept in sterile con-
tainers with sterilized soil and water for 17 days in July, 2018. This time-frame, long enough
for beetles to feed 5–6 times, was chosen to examine short-term dietary impacts. Banana-fed
(Trader Joe’s, Dole Banana Ecuador) and mealworm-fed (Timberline, Vita-bugs Mini Meal-
worms 500 count) beetles were fed on the first day, and subsequently fed and watered every
three days using heat-sterilized forceps and autoclaved water. All feeding portions consisted of
0.04g (+/- 0.01g) non-sterile food. Banana and mealworm bacterial communities were
sequenced as controls and were removed from the analysis after confirming samples were not
contaminated. Starved beetles received water, but no food. On the last day, beetles were quickly
anesthetized by placing them for one minute at -80˚C in their plastic containers. All speci-
mens, including wild-caught beetles, were dissected as described by McManus et al. [31]. Each
beetle was dissected into three groups of tissues: secretory cells, gut (including foregut, midgut,
and hindgut), and the rest of the body minus the secretory cells and gut (subsequently referred
to as ‘partial body’). Parasitic worms (Nematomorpha) found to be infecting one starved beetle
and one mealworm-fed beetle were removed from those specimens and the worm tissues not
included in downstream analysis.
DNA extraction, PCR, and next generation sequencing
Tissues were incubated overnight in a 9:1 ratio of buffer ATL and proteinase K (Qiagen
DNeasy Blood & Tissue Kit) at 55˚C on a rocking tray. Lysate from overnight incubation was
transferred to sterile 1.5ml O-ring tubes containing 0.25g (+/- 0.02g) of 0.1mm diameter zirco-
nium beads and bead beat at 2000rpm for 3 minutes in a PowerLyzer to lyse bacterial cells.
DNA was extracted from the lysed homogenate using Solid Phase Reversible Immobilization
(SPRI) magnetic beads made following the method of Rohland [32]: 100μL lysate was mixed
with 180μL of well-mixed, room temperature SPRI beads, incubated for approximately 5 min-
utes on the bench, then transferred to a magnetic rack. After the SPRI beads pelleted, 200μL
80% ethanol was added. After 30 seconds the supernatant was removed, the ethanol wash was
repeated a second time and the supernatant was removed again. Then, the tubes containing
SPRI bead tubes were removed from the magnetic rack and allowed to air dry completely.
DNA was eluted by adding 50μL TB solution (10mM Tris) directly onto the beads and incu-
bating for 5 minutes, then returning samples to the magnetic rack to pellet the SPRI beads and
retrieve the DNA-containing supernatant.
The V4 region of the 16S rRNA gene was PCR amplified in duplicate in 25μL reactions
using GoTaq Green Master Mix (Promega), and the resulting PCR products were subsequently
pooled. During the first round, previously described primers [33] 515FB_in (5’-ACA CTC
TTT CCC TAC ACG ACG CTC TTC CGA TCT GTG YCA GCM GCC GCG GTA A-3’)
and 806RB_in (5’-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC TGG ACT
ACH VGG GTW TCT AAT-3’), which were adapted to be complementary to the second
round primers [34], were added to the ends of all 16S genes with the following conditions
(BioRad thermocycler): initial denaturation at 94˚C for 3 min, followed by 30 cycles of 94˚C
PLOS ONE
Host diet and microbial diversity in ground beetles
PLOS ONE | https://doi.org/10.1371/journal.pone.0241529 March 19, 2021 4 / 16

for 45 sec, 50˚C for 1 min, 72˚C for 1:30 min, and a final extension step of 72˚C for 10 min. A
second round of PCR was performed using unique combinations of barcoded forward (5’-
AAT GAT ACG GCG ACC ACC GAG ATC TAC ACX XXX XXX XAC ACT CTT TCC
CTA CAC GA-3’) and reverse (5’-CAA GCA GAA GAC GGC ATA CGA GAT XXX XXX
XXG TGA CTG GAG TTC AGA CGT G-3’) primers [34] to create a dual-index amplicon
library for Illumina sequencing (position of barcodes indicated by ’X’ characters). The condi-
tions for the second PCR reaction were: initial denaturation at 94˚C for 3 min, followed by 10
cycles of 94˚C for 45 sec, 50˚C for 1 min, 72˚C for 1:30 min, and a final extension step of 72˚C
for 10 min. All pooled duplicate PCR products were run on a 1% agarose gel for 30 min at
100V, and imaged under UV light to verify successful PCR. DNA concentration was quantified
using a Qubit fluorometer, and equimolar amounts were pooled. The pooled library was puri-
fied (Qiagen Qiaquick PCR Purification Kit) and sent for Illumina MiSeq sequencing at the
U.C. Berkeley Genomics Sequencing Laboratory.
Analysis
Amplicon reads for the V4 region of 16S were de-multiplexed with deML [35] and processed
using DADA2 [36], including quality filtering with maxEE = 2. Reads were de-replicated into
unique 16S amplicon sequence variants (ASVs, also referred to as phylotypes) using a read error
model parameterized from the data. Paired-end reads were merged and mapped to ASVs to con-
struct a sequence table. Chimeric sequences were removed. Taxonomic assignments for exact
matches of ASVs and reference strains were made using the Ribosomal Database Project database
[37]. Sequence tables and taxonomic assignments were imported into R version 3.5 [38] for
downstream analysis and combined into a single phyloseq [39] object for convenience. To
account for variation in sequencing effort across samples, samples were scaled according to vari-
ance stabilized ASV abundances using DESeq2 [40, 41]. ASV alignments made using DECIPHER
[42, 43] were used to construct a neighbor-joining tree, and this tree was then used as the starting
point for deriving a maximum likelihood tree from a generalized time-reversible model with
gamma rate variation, implemented with the phangorn package in R [44]. The tree was rooted
using QsRutils [45]. For comparative analysis between beetles, ASV data from all three tissues of
each specimen were combined into an aggregate bacterial community. Alpha diversity measures
were calculated using the packages phyloseq [39] and picante [46]. Non-metric multidimensional
scaling (NMDS) plots of beta diversity were created using phyloseq [39], and analysis of similari-
ties (ANOSIM) tests were run using the package vegan [47]. Bray-Curtis distances were calcu-
lated both for aggregate community data and for the original dataset. Venn diagrams of
phylotypes present by diet were rendered by VennDiagram [48]. To control for possible sequenc-
ing errors, only phylotypes occurring at least twice in the entire dataset were included in venn
diagram analysis. Hierarchical clustering of communities was performed with the package ape
[49]. Secretory cells were tested for differential abundance of microbe phylotypes using an equiv-
alent method to RNA-seq differential expression analysis, implemented using DESeq2 [39, 40].
Ethics statement
No permits were required for the described study, which complied with all relevant regulations.
Results
Sequencing results
After quality filtering, the mean number of reads per sample was 19,868, and the median num-
ber of reads per sample was 17,532.
PLOS ONE
Host diet and microbial diversity in ground beetles
PLOS ONE | https://doi.org/10.1371/journal.pone.0241529 March 19, 2021 5 / 16

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Journal ArticleDOI
TL;DR: In this article , the authors identify the main influences on microbiome composition in a specialist insect, the western corn rootworm (WCR), by analyzing the bacterial communities of adults collected from their obligate host plant, corn (Zea mays), across several geographic locations and comparing the patterns in communities to its congeneric species, the northern corn rootworms (NCR; Diabrotica barberi).
Abstract: Microbial communities associated with animals vary based on both intrinsic and extrinsic factors. Of many possible determinants affecting microbiome composition, host phylogeny, host diet, and local environment are the most important. How these factors interact across spatial scales is not well understood. Here, we seek to identify the main influences on microbiome composition in a specialist insect, the western corn rootworm (WCR; Diabrotica virgifera virgifera), by analyzing the bacterial communities of adults collected from their obligate host plant, corn (Zea mays), across several geographic locations and comparing the patterns in communities to its congeneric species, the northern corn rootworm (NCR; Diabrotica barberi). We found that bacterial communities of WCR and NCR shared a portion of their bacterial communities even when collected from disparate locations. However, within each species, the location of collection significantly influenced the composition of their microbiome. Correlations of geographic distance between sites with WCR bacterial community composition revealed different patterns at different spatial scales. Community similarity decreased with increased geographic distance at smaller spatial scales (~25 km between the nearest sites). At broad spatial scales (>200 km), community composition was not correlated with distances between sites, but instead reflected the historical invasion path of WCR across the United States. These results suggest bacterial communities are structured directly by dispersal dynamics at small, regional spatial scales, while landscape-level genetic or environmental differences may drive community composition across broad spatial scales in this specialist insect.
Journal ArticleDOI
29 Nov 2022-Insects
TL;DR: In this paper , the authors used the Chlaenius pallipes (Carabid beetle) as a model organism from the terraced paddy fields and the large-sized paddy field.
Abstract: Simple Summary This study aimed to determine i the gut microbiome could be used as an indicator of an organism’s health or the health of the population using the Chlaenius pallipes (Carabid beetle) as a model organism from the terraced paddy fields and the large-sized paddy fields. The composition of the gut bacterial community associated with habitat types. Gut bacterial community of C. pallipes differed in diversity, similarity, and composition between the habitat conditions. Additionally, food resource quality derived from the stable nitrogen isotope ratio was significantly correlated with richness of the gut bacterial community. Abstract We investigated whether the gut bacterial community of Chlaenius pallipes could represent the health conditions of individuals or populations based on where these beetles inhabit. Considering the ecological traits of the species, the gut bacterial communities of carabid populations inhabiting stable or unstable habitats were compared. Food resource quality (δ15N) and morphological shape, especially body and wing size, may be significant factors that directly or indirectly affect the gut bacterial community of carabid beetles. Firmicutes (51.7%) and Proteobacteria (36.3%) were the predominant phyla in the gut bacterial community of C. pallipes. A significant difference in the gut bacterial community structure was observed between organisms inhabiting unstable and stable habitats in this study. Wing size, as estimated by centroid size, was correlated with differences in the gut bacterial community composition of the species. Even if a factor is not strong enough to determine the survival of carabid beetles, the composition of the gut bacterial community can change. We found that although each individual has a large variation in the gut bacterial community composition, the gut bacterial community can be used to evaluate the condition of each habitat through consistent investigation. Habitat assessment based on changes in the number of carabid beetle species and their composition requires relatively long-term research; however, the gut bacterial community of carabid beetles can help identify short-term environmental changes.
References
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Journal Article
TL;DR: Copyright (©) 1999–2012 R Foundation for Statistical Computing; permission is granted to make and distribute verbatim copies of this manual provided the copyright notice and permission notice are preserved on all copies.
Abstract: Copyright (©) 1999–2012 R Foundation for Statistical Computing. Permission is granted to make and distribute verbatim copies of this manual provided the copyright notice and this permission notice are preserved on all copies. Permission is granted to copy and distribute modified versions of this manual under the conditions for verbatim copying, provided that the entire resulting derived work is distributed under the terms of a permission notice identical to this one. Permission is granted to copy and distribute translations of this manual into another language, under the above conditions for modified versions, except that this permission notice may be stated in a translation approved by the R Core Team.

272,030 citations

Journal ArticleDOI
TL;DR: This work presents DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates, which enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression.
Abstract: In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html .

47,038 citations

Journal ArticleDOI
TL;DR: The open-source software package DADA2 for modeling and correcting Illumina-sequenced amplicon errors is presented, revealing a diversity of previously undetected Lactobacillus crispatus variants.
Abstract: We present the open-source software package DADA2 for modeling and correcting Illumina-sequenced amplicon errors (https://github.com/benjjneb/dada2). DADA2 infers sample sequences exactly and resolves differences of as little as 1 nucleotide. In several mock communities, DADA2 identified more real variants and output fewer spurious sequences than other methods. We applied DADA2 to vaginal samples from a cohort of pregnant women, revealing a diversity of previously undetected Lactobacillus crispatus variants.

14,505 citations

Journal ArticleDOI
22 Apr 2013-PLOS ONE
TL;DR: The phyloseq project for R is a new open-source software package dedicated to the object-oriented representation and analysis of microbiome census data in R, which supports importing data from a variety of common formats, as well as many analysis techniques.
Abstract: Background The analysis of microbial communities through DNA sequencing brings many challenges: the integration of different types of data with methods from ecology, genetics, phylogenetics, multivariate statistics, visualization and testing. With the increased breadth of experimental designs now being pursued, project-specific statistical analyses are often needed, and these analyses are often difficult (or impossible) for peer researchers to independently reproduce. The vast majority of the requisite tools for performing these analyses reproducibly are already implemented in R and its extensions (packages), but with limited support for high throughput microbiome census data. Results Here we describe a software project, phyloseq, dedicated to the object-oriented representation and analysis of microbiome census data in R. It supports importing data from a variety of common formats, as well as many analysis techniques. These include calibration, filtering, subsetting, agglomeration, multi-table comparisons, diversity analysis, parallelized Fast UniFrac, ordination methods, and production of publication-quality graphics; all in a manner that is easy to document, share, and modify. We show how to apply functions from other R packages to phyloseq-represented data, illustrating the availability of a large number of open source analysis techniques. We discuss the use of phyloseq with tools for reproducible research, a practice common in other fields but still rare in the analysis of highly parallel microbiome census data. We have made available all of the materials necessary to completely reproduce the analysis and figures included in this article, an example of best practices for reproducible research. Conclusions The phyloseq project for R is a new open-source software package, freely available on the web from both GitHub and Bioconductor.

11,272 citations

Journal ArticleDOI
TL;DR: It is shown that the protocol developed for these instruments successfully recaptures known biological results, and additionally that biological conclusions are consistent across sequencing platforms (the HiSeq2000 versus the MiSeq) and across the sequenced regions of amplicons.
Abstract: DNA sequencing continues to decrease in cost with the Illumina HiSeq2000 generating up to 600 Gb of paired-end 100 base reads in a ten-day run. Here we present a protocol for community amplicon sequencing on the HiSeq2000 and MiSeq Illumina platforms, and apply that protocol to sequence 24 microbial communities from host-associated and free-living environments. A critical question as more sequencing platforms become available is whether biological conclusions derived on one platform are consistent with what would be derived on a different platform. We show that the protocol developed for these instruments successfully recaptures known biological results, and additionally that biological conclusions are consistent across sequencing platforms (the HiSeq2000 versus the MiSeq) and across the sequenced regions of amplicons.

6,840 citations

Frequently Asked Questions (2)
Q1. What are the contributions mentioned in the paper "Persistence of the ground beetle (coleoptera: carabidae) microbiome to diet manipulation" ?

Here the authors examine the relationship between host environment, host traits, and microbial diversity in three species in the ground beetle family ( Coleoptera: Carabidae ), a group of roughly 40,000 species that synthesize a wide diversity of defensive compounds. This study used 16S amplicon sequencing to profile three species that are phylogenetically distantly related, trophically distinct, and whose defensive chemical secretions differ: Anisodactylus similis LeConte, 1851, Pterostichus serripes ( LeConte, 1875 ), and Brachinus elongatulus Chaudoir, 1876. 

Future studies could investigate these and other factors that may affect carabid microbiomes throughout the carabid life cycle. As a baseline for future studies of carabid secretory cell microbes to build upon, the authors determined that several bacterial genera in particular ( Flavobacterium and an unknown Comamonadaceae genus ) are differentially abundant in the secretory cells over other tissues. In addition to more thorough investigations of secretory cell microbiome composition across Carabidae, the authors propose that future studies should directly test the possibility that a symbiotic relationship with microbes plays a role in host chemical biosynthesis. Their preliminary study found that the pygidial glands possess a differentiated microbiome from other carabid tissues, warranting further investigation especially given the unique metabolic capabilities of these cells.