Session 4: Resource Development and Techniques
Handling and Analysis of Multi-dimensional Imaging Data
Carsten Wolff
Marine Biological Laboratory
Abstract
This presentation will provide a general overview of current imaging modalities that allow long-term observation of arthropod development in a high spatial and temporal resolution. One of the biggest challenges still is the handling of multi-terabyte datasets and how to extract biologically meaningful information from it. I will present and hopefully demystify all required steps of the processing pipeline that are needed to successfully register multi-dimensional datasets. Furthermore, I will discuss routes of how to approach subsequent image analysis and how to visualize the data.
Session
Resource Development and Techniques
in situ HCR in non-traditional arthropods
Heather Bruce
Marine Biological Laboratory
Abstract
Visualizing the expression of genes is a fundamental tool in molecular biology. Traditional colorimetric in situ hybridization using long RNA probes has been a staple for visualizing gene expression but has many drawbacks. In situ HCR v3.0, developed by Choi et. al. 2018, offers improvements over traditional in situs in nearly every aspect: probes can simply be ordered rather than painstakingly cloned and transcribed, which also makes them cost-effective; an HCR takes just three days to complete rather than five or more days; HCR is robust and works well for first-time users; and HCR probes can be multiplexed, allowing four to eight genes to be visualized in a single sample. HCR has been used successfully in many arthropods, including insects (Drosophila, Tribolium), crustaceans (Parhyale, Daphnia, Artemia), and chelicerates (Limulus horseshoe crab, Acanthoscurria tarantula). In this demo, you will learn how to design and order HCR probes as well as best practices for experimental design. Please bring your laptop.
Session
Resource Development and Techniques
An efficient and precise gene knock-in strategy with modified DNA donors in emerging model insects
Taro Nakamura1,2, Toshiya Ando1,2,3,4, Yuji Matsuoka1 and Teruyuki Niimi1,2
1Division of Evolutionary Developmental Biology, National Institute for Basic Biology, Okazaki, Aichi 444-8585, JAPAN; 2 Basic Biology Program, SOKENDAI, Okazaki, Aichi 444-8585, JAPAN; 3JST, PRESTO, Kawaguchi, Saitama 332-0012, JAPAN;
4the Hakubi Center for Advanced Research, Kyoto University, Kyoto, Kyoto 606-8501, JAPAN
Abstract
The CRISPR-Cas9 genome editing technology has opened new possibilities of genetic research in various emerging model insects. However, due to the technical challenges associated with establishing knock-in strains, applications in these insects are primarily limited to knockout experiments. A major obstacle in achieving successful knock-in experiments is the markedly low insertion efficiency into the target gene. Here, we tested multiple knock-in strategies in the harlequin ladybird Harmonia axyridis and the two-spotted cricket Gryllus bimacualtus, emerging model insects for evolutionary developmental studies. We found that ssDNA templates efficiently generated founder knock-in strains (10-20%), while the 5’ region of ssDNA templates was frequently trimmed when insert lengths exceeded ~40 bases. Based on these findings, we investigated various DNA templates with modifications to the 3’ terminal region and discovered a high founder generation efficiency (20-65%) with high accuracy. This approach is an effective method for generating knock-in strains in emerging model insects for developmental genetic research.
Session
Resource Development and Techniques
DIPA-CRISPR Gene Editing in Insects
Yu Shirai and Takaaki Daimon
Kyoto University
Abstract
With over a million species described, insects are a treasure trove of diversity and represent boundless possibilities as research tools for answering fundamental questions in biology.
Current approaches for gene editing in insects require microinjection of materials into early embryos, which is highly challenging. To overcome these limitations, we have recently developed a method named “direct parental CRISPR (DIPA-CRISPR)”, which enables highly efficient gene editing by injecting Cas9 ribonucleoproteins (RNPs) into adults.
In the DIPA-CRISPR method, Cas9 RNPs are injected into the body cavity of an adult female undergoing vitellogenesis (generating eggs in the yolk-uptake phase of oogenesis), and the female produces gene-edited offspring. Commercial Cas9 protein can be directly used for DIPA-CRISPR, making this method simple and straightforward. In our hands, more than 50% (in the beetle Tribolium castaneum) or 20% (in the cockroach Blattella germanica) of G0 offspring became gene-edited individuals under optimized conditions. In addition, we have recently shown that DIPA-CRISPR can be applied to mosquitoes (Aedes aegypti) and hemipteran bugs (Plautia stali and Orius strigicollis). In principle, its application could be extended to other arthropods such as chelicerates (mites and spiders) and crustaceans (shrimp and crabs).
The original DIPA-CRISPR method uses a commercial Cas9, which hinders its application in large species due to the increasing cost of reagents. Therefore, we optimized and established a procedure for Cas9 purification. The performance of our in-house Cas9 was comparable to that of commercial Cas9, solving the cost issue and paving the way for rational engineering of Cas9 to further increase the efficiency and versatility of the DIPA-CRISPR approach.
In this talk, I would like to introduce and share the latest updates of the DIPA-CRISPR method.
Session
Resource Development and Techniques
Non-traditional insect models are key for understanding the evolution and regulation of insect metamorphosis
Guillem Ylla
Jagiellonian University
Abstract
Holometabolous metamorphosis is one of the most successful innovations of insects, currently present in 90% of the living insect species. This development type emerged from the incomplete or hemimetabolous metamorphosis, which, in turn, evolved from the ancestral direct ametabolous development. Because most of the typical insect models are holometabolous, data on species that preserved these ancestral developmental types is scarce, yet is essential to understand the evolution of insect metamorphosis and its regulation. Thus, in the last few years, we have put significant efforts into sequencing new genomes of hemimetabolous species and releasing comprehensive transcriptomic datasets.
Thanks to those efforts, we are now in a position to start leveraging these data to shed light on how metamorphosis is regulated, emerged, and evolved. For instance, this approach allowed us to identify a new role of E93 in the embryo which could have been instrumental in the emergence of insect metamorphosis. Initially, comparative transcriptomics along the ontogeny of the hemimetabolous Blattella germanica and the holometabolous Drosophila melanogaster, allowed us to identify several transcriptomic differences that could have played a role in the evolution of insect metamorphosis. Notably, the expression pattern of E93, the factor that triggers metamorphosis, was remarkably different. In addition to the canonical pre-adult expression peak, in B. germanica E93 appeared expressed in the early embryo. Depletion of E93 transcripts in B. germanica embryos interrupted the development and triggered the downregulation of genes involved in development. Then, querying hundreds of publicly available transcriptomic data from insect embryos and preadults, we tested whether high and low E93 levels in the embryo could be a general trend in hemimetabolans and holometabolans respectively.
Thanks to the publicly available genomes and transcriptomes of dozens of non-traditional insect models, we were able to determine that E93 expression is high in the embryos of ametabolans and hemimetabolans while low in holometabolan embryos. This led us to hypothesize that E93 in the embryo might be responsible for the development of adult-like juveniles, and that the reduction of embryonic expression of E93 could have been instrumental in the emergence of the larva, and hence, for the emergence of the holometabolous metamorphosis.
Session
Resource Development and Techniques