Leader: Ewald Grosse-Wilde

Here we focus on the bark beetle itself. The goal is to find new ways to intervene in the mass devastation
of forests by looking at two specific parts of the beetle lifestyle: How it coordinates the mass attacks used
to overcome tree defences, and how it is adapted to feeding on the trees. To this end we investigate
the communication and host selection via pheromones and host volatiles that the beetles use in coordinating
mass attacks, as well as try to identify genes and enzymes that deal with tree defences.

Tasks G1

During our initial years we have sequenced and analysed the genome of our main species of interest,
the Eursian Spruce Bark Beetle Ips typographus. The genome database as well as previous publication
on olfactory receptor genes in the species have been used for the identification of suitable target genes
in G1a. Simultaneously we have established a pipeline for the generation of dew flies (Drosophila melanogaster)
that express bark beetle receptors in their antennae to allow for functional analysis of the receptors proteins
in a controlled environment, using single sensillum recordings (G1b). This will allow us to associate receptors
with the detection of specific odorants of importance in the context of beetle ecology.

Task G1a: Genomes of several bark beetle species, including the Eurasian spruce bark beetle Ips typographus

Task leader: Ewald Grosse-Wilde
Task members: Amit Roy, Jibin Johny

HIGHLIGHT:

Powell D, Große-Wilde E, Krokene P, Roy A, Chakraborty A, Löfstedt C, Vogel H, Andersson MN, Schlyter F. 2021. A highly contiguous genome assembly of a major forest pest, the Eurasian spruce bark beetle Ips typographus. Communications Biology 4: 1059. 10.1038/s42003-021-02602-3

“De-orphanisation” by Empty Neuron System principles from (Gonzales et al., 2016) SSR setup. Left) Overview with computer. Right) Close-up; microscope, recording electrodes.

Task G1b:De-orphanisation of bark beetle Olfactory Receptors

Task leader: Dr. Blanka Kalinova
Task members: Dr. Souleymane Diallo, Ali Qasim, Jibin Johny,

The olfactory system mediates detection of odours in the environment and has direct influence on host plant selection. We use the genome and other sources on gene content of the beetle to identify so called Olfactory Receptors (ORs). Insect ORs are generally used in insects to identify chemical cues, and especially in the detection of volatile, long-distance pheromones and plant compounds. In close collaboration with our partners in EXTEMIT-K we will try to identify specific ORs in the bark beetle as new targets for intervention. A long-term goal is to find compounds that block such receptors, which would essentially negate the pheromone and severely reduce mass attacks. The first step, in which the correct receptors are identified (De-orphanisation), is done by manipulating vinegar flies (Drosophila melanogaster) to express beetle receptors. Here we make use of the so-called Empty Neuron System (ENS) that was originally established by Hallem and Carlsson (2004) (Figure at right). Flies will express the bark beetle receptor genes in specific neurons on their antennae that lack a fly OR. The flies can then be tested in electrophysiological experiments, directly measuring the activity of the neuron when stimulated with odours in so called Single Sensillum Recordings (SSR, below). If the receptor is a pheromone receptor, the neuron will respond to bark beetle pheromone. This approach has been used many Diptera and a few Lepidoptera (de Fouchier et al., 2016), we attempt the first in Coleoptera on large scale by ENS method (Empty Neuron System).

Task G1: Progress and Outlook

Our high-quality genome database by sequencing DNA by Pac Bio from highly inbred beetle strains is published (Powell et al. 2021). This dataset will not only be used to support EXTEMIT-K work but is also publicly available, prepared to be useful for other scientists working with Ips species and other bark beetles.

Beyond serving as a foundation for the other projects in G-level, we are currently working on expanding genome sequencing efforts to other Ips species with different host profiles, in an effort to see which genes/gene families are primarily affected by this lifestyle change. This project is the basis of several collaborations, and also includes members of G2.

The well-staffed rearing facility for dew flies (Drosophila melanogaster) and a pipeline for the generation of fly line that express bark beetle receptors in their antennae to allow for functional analysis of the receptors proteins in a controlled environment, using single sensillum recordings (G1b). This will allow us to associate receptors with the detection of specific odorants of importance in the context of mass attacks on spruce forests.

Additionally, we can combine the physiological tests with gas chromatography (GC) to identify new, specialized receptors that detect so far unknown compounds that might be important for the beetle (Figure at right). This allows us to test many volatile compounds from sources of material potentially bad for the beetle, like old, colonised bark, and determine which compound (GC) activates which receptor in a sensillum (SSR). Using this approach, we can find novel compounds that are of ecological relevance to the beetle, and directly associate them with the responding OR.

De-orphanisation & GC-SSR-ENS: (Gas Chromatography – Single Sensilllum Recording – Empty Neuron System): Allows analysis by stimulation of single compound from complex samples directly to defined ORs.

Task G2: Molecular underpinnings of plant-beetle interaction

Task leader: Dr. Amit Roy
Task members: Dr Gothandapani Sellamuthu, Muhammad Zubair Ashraf, Mallikarjuna Reddy JOGA,
Aisha Naseer, Arunabha Khara

Under G2 task at Forest Molecular Entomology Lab (photos above), we are trying to disentangle the
bark beetle adaptation against the tree defences under stresses in the forest using state-of-the-art
metagenomics (Chakraborty et al. 2020a, 2020b), transcriptomic (Sellamuthu et al 2021),
and proteomic approaches.

HIGHLIGHTS:

Chakraborty A, Ashraf MZ, Modlinger R, Synek J, Schlyter F, Roy A. 2020a. Unravelling the
gut bacteriome of Ips (Coleoptera: Curculionidae: Scolytinae): identifying core bacterial
assemblage and their ecological relevance.Scientific Reports 10: 1-17 10.1038/s41598-020-75203-5

Chakraborty A, Modlinger R, Ashraf MZ, Synek J, Schlyter F, Roy A. 2020b. Core mycobiome
and their ecological relevance in the gut of five Ips bark beetles (Coleoptera: Curculionidae: Scolytinae).
Frontiers in Microbiology 11: 2134 10.3389/fmicb.2020.568853

Sellamuthu G, Amin S, Bílý J, Synek J, Modlinger R, Sen MK, Chakraborty A, Roy A, 2021.
Reference gene selection for normalizing gene expression in Ips sexdentatus
(Coleoptera: Curculionidae: Scolytinae) under different experimental conditions. Frontiers in Physiology, (Accepted).

Task G2a: Molecular mechanisms underlying bark beetle resistance

against Conifer defence

Task members: Dr. Amit Roy, Gothandapani Sellamuthu, Muhammad Zubair Ashraf,
Aisha Naseer, Mallikarjuna Reddy JOGA.

To compete, overcome, or outwit opponents, organisms have evolved elaborate “toxins.” Beetles are not an exception to that. Precisely, bark beetles are eruptive forest insects that have the potential to cause landscape-level mortality to conifer forests. Conifers possess formidable defences, which bark beetles must overcome to reproduce and thrive. Hence, it is of utmost importance to know how these tiny beetles such as Ips typographus overcome the formidable defences of spruce at difference stress levels. Hence, under this project, we are going to delineate the digestion and detoxification of host plant toxins in the midgut of the bark beetle (i.e., Ips typographus) via transcriptomics and proteomics approach. We will also extend our understanding of the given topic by using the RNAi approach.

Task G2 b: Understanding the microbial contribution in bark beetle adaptation to host plants.

Task members: Dr. Amit Roy, Arunabha Khara

Coniferous trees can produce defensive chemicals, such as monoterpenes, in defence against bark beetle attacks. Gut bacteria of bark beetles were reported to possess many beneficial ecological functions, including degradation of defensive chemicals and nutrition provision. Previous studies described a complex and elusive interaction between gut microbiota of beetles and their host. Although gut bacterial communities of bark beetles at different life stages had been investigated, no research had been conducted on different Ips species such as Ips typographus, Ips cembrae, Ips duplicatus, etc. on this crucial and ecologically significant aspect related to insect adaptive physiology. Under the present project, we would like to disentangle the gut microbial contribution to bark beetle adaptive physiology.

Primary collaborators for G2:

Dr. Massimo Faccoli, University of Padova, Italy
Dr. Fredrik Lavander, Lund University, Sweden
Prof. Lynne K. Rieske-Kinney, University of Kentucky, USA
Prof. Guy Smagghe, Ghent University, Belgium

Task G2: Progress and Outlook

At present, we are carrying out gene expression studies and microbial studies further to enhance our understanding
on the beetle adaptation to host and climate change, which may facilitate formulating better pest management
strategy in future.

Recently, we have identified reference genes in Ips typographus (Sellamuthu et al 2021), to facilitate ongoing
gene expression studies in our group and others working on the species worldwide. We have identified for the
very first-time the core microbial assemblage in five Ips bark beetle gut obtained from Czech forests and try
to evaluate their ecological relevance from scratch (Chakraborty et al. 2020a, 2020b).

The goal is to find new intervention targets, but also to couple the beetle response to findings from T-level
on new semiochemicals that play a role in beetle-tree interactions.

A new avenue added, partly mentioned above, is RNA interference (RNAi) for both research and management.
RNAi allows inhibition of translation of nuclear DNA (genes) via mRNA to proteins for metabolic processes
essential for fitness such as growth and reproduction. Management for pest control is a future option now
becoming possible for the cryptic wood boring beetles, such as bark beetles (Reddy et al. 2021).

HIGHLIGHT:

Reddy M, Mogilicherla K, Smagghe G, Roy A, 2021. RNAi against wood-boring forest pests RNA
interference-based forest protection products (FPPs) against wood-boring coleopterans: hope or hype?
Frontiers in Plant Science, 1943. doi:10.3389/fpls.2021.733608.

For other G-level publications, please see the EXTEMIT-K summary of publications per year