Individual spotted handfish can be identified based on their unique spot patterns Credit: © Carlie Devine
Scientists trying to save the spotted handfish refer to it as an “uglycute” little fish. At about 5 to 12 centimetres long, its most prominent features include human-hand-shaped fins and a lure dangling above its mouth – all the better to nab tiny prey.
In 1996, the spotted handfish (Brachionichthys hirsutus) nabbed the dubious distinction of first marine fish to be declared critically endangered.
Today, conservation biologists at CSIRO, Australia’s national science agency, estimate only 2,000 individuals remain in their native waters off southeast Tasmania.
Fortunately, the long, ongoing fight to reverse the spotted handfish’s plight got a boost from cutting-edge technology in genomics, the field of molecular biology focused on mapping and characterising the entire set of genetic material in an organism.
Last October, CSIRO’s Applied Genomics Initiative (AGI) announced that they, along with collaborators at the Australian National University’s Biomolecular Resource Facility (ANU-BRF), sequenced the entire spotted handfish genome.
The genetic sequence – all 406,634,837 nucleotide “letters” long – is publicly available on the US-based National Center for Biotechnology Information database.
“It’s a big leap forward in conservation,” says Carlie Devine, a research technician with CSIRO’s Threatened, Endangered or Protected Species team.
Eventually the CSIRO-led teams aim to use the genome for moonshot projects like forecasts of the handfish’s climate resilience.
“Essentially this means that the genome will be able to tell us the capacity the species has for change in response to climate pressures,” explains Devine.
But the path towards these goals was not without challenges, and hurdles for using the technology remain.
Spotted handfish use their hand-like fins to ‘walk’ along the seafloor. Credit: © Rick Stuart-Smith
Piecing together a difficult genome
A genome is the complete sequence of DNA nucleotide bases found inside virtually every cell of an individual. The pattern of nucleotide bases includes the instructions – the genes – for all the proteins that make up a cell and its metabolism.
The AGI team got their chance to sequence the spotted handfish genome when a captive individual died of natural causes. Tom Walsh, a principal research scientist and co-lead at AGI, and Leon Court, a senior experimental scientist at AGI spoke to Cosmos about the intensive process.
Sequencing a genome usually involves amplifying the DNA of interest, tagging the new DNA with fluorescent proteins unique to each nucleotide letter, and reading the resulting pattern of colours.
The CSIRO-led team ran into difficulties with the first step: amplifying the handfish DNA. Marine samples are notoriously challenging because “you often get a whole lot of contaminating bacteria and fungus,” says Walsh. “Especially from a dead sample.”
To make matters worse, fish samples often contain inhibitors, proteins that block the DNA polymerase enzymes from amplifying the DNA.
“With the sequencing process, it’s often an exploration, so you try one approach and then if it’s not working then you pivot. So we tried it with the DNA that we extracted from the fish,” says Court.
In time, the team were able to extract what looked like enough DNA, “but we could tell … that it wasn’t very good quality,” says Walsh.
To address this issue, the CSIRO team partnered with ANU-BRF to try a low input protocol technique. The protocol is designed to amplify small amounts of quality DNA, such as samples from tiny organisms, like a single mosquito.
The low input protocol relies on relatively recent technology and only a handful of facilities around the world are set up to do it. CSIRO and ANU-BRF are early adopters, and they have developed workflows to handle difficult samples.
Central to the protocol are DNA polymerase enzymes with high fidelity and high processivity, which means they accurately copy long lengths of the fish’s native DNA before falling off.
“The freshly synthesised ‘copied DNA’ is undamaged and lacks the troublesome modifications and inhibitors, and so can be sequenced with a much higher efficiency than the original ‘native DNA’,” says Court.
Generating long strands of quality DNA is important for the next step: long-read sequencing. Unlike traditional short-read sequencing which generates billions of ‘reads’, each about 150 nucleotides in length, long-read sequencing generates ‘reads’ up to 19,000 nucleotides long.
“One of the powerful features of highly accurate long-reads is they’re easier to assemble genomes from bioinformatically because you’re working with larger jigsaw pieces rather than many smaller ones,” explains Court.
Because of the larger overlaps between segments, long-read sequencing has led to fewer gaps in assembled genomes. This makes the newly completed spotted handfish reference genome particularly high-quality.
The CSIRO AGI and ANU-BRF teams preparing samples for their new PacBio Revio sequencer. Credit: Millennium Science
A digital transformation of biology
“Every genome is the start of an impact journey,” says Walsh.
Notwithstanding the challenges of sequencing the spotted handfish genome, “generating the data is not the problem anymore, it’s the analysis of it,” says Walsh. “And the decisions that it can help inform, so the utility of genomic information is really harnessed by decision makers,” adds Court.
Walsh says the utility of genomic information starts with annotating the genome, by “saying where the genes are and what they are [for]”.
The AGI team aims to integrate all the available information molecules, from DNA to proteins, to ground-truth the annotations and better understand an organism. (Study of complete sets of information molecules is often referred to as “-omics” and it includes genomics, transcriptomics, and proteomics for DNA, RNA, and proteins, respectively.)
For example, transcriptomes of messenger RNA, which is directly transcribed from DNA, can validate gene designations in the genome.
“Similarly, knowledge of proteins allows you to validate which genes are actually being expressed and transcribed,” says Court.
Walsh says a next possible step is “modelling the protein and trying to understand the function and then coming back into the lab and testing this function”.
Integrating -omics datasets has many applications beyond conservation too. AGI is pursuing projects across biosecurity, health, agriculture and ecotoxicology. For example, identifying genes that encode proteins that make crop pests resistant to pesticides.
Relevant to the handfish, the function of proteins becomes important when trying to assess the climate adaptability of a species.
“Things like climate or thermal tolerance, they are very unlikely to be a single change in the genome,” says Walsh. “They’re much more likely to be a suite of complicated changes.”
The new genome holds a lot of potential but as Walsh points out, “genomes will not save species. They are tools that can be used to help.”
The team’s sequencer, open and ready to accept samples and reagents.
Credit: CSIRO AGI
Handfish conservation, present and future
As a tool, the genome comes at a precarious time for the spotted handfish.
When asked about the fish’s outlook, Devine is matter of fact: “It’s definitely in decline. Each site’s got a downward trend. That’s why it’s critically endangered.”
Currently the CSIRO TEPS team monitors 9 sites with isolated populations of spotted handfish in Tasmania’s Derwent Estuary. This involves time-consuming and expensive SCUBA dives where scientists monitor habitats and population size as well as collect small skin clips for genetic information.
Devine says they will use the handfish genome information for future studies and to help with population monitoring. “That would probably be our first step forward.”
One low-cost population monitoring technique made possible by the sequenced genome is environmental DNA (eDNA). eDNA refers to fragments of DNA floating in the water column which reflect the diversity of living things nearby. It can be collected simply, and handfish DNA fragments could be matched to the reference genome.
“We might be able to find little pockets where handfish are doing well and that we haven’t SCUBA-dived,” says Devine.
The genome could also inform CSIRO’s captive breeding program – in terms of matching up parents and releasing their offspring into wild populations – all to increase spotted handfish genetic diversity.
Genetic diversity provides the variation for natural selection to act on, and this will become increasingly important as Tasmania’s waters warm.
In the near future, the CSIRO teams hope to use the genome and the integrated “-omics” information to assess the handfish’s climate resilience.
For example, proteomics could provide “protein information for the role of adaptive molecules like ‘heat shock’ proteins… in a species,” says Court.
Additional -omics information on lipids and metabolites could help the teams characterise individuals that are heat stressed and how they differ from non-stressed individuals.
Another viable avenue is to sequence more genomes from more individuals representing different sites.
“Pan-genomics would give us a better handle on the species genetic ability to adapt to climate change,” says Court. “This is something AGI is well placed to do.”
Despite the challenges facing the spotted handfish, the CSIRO teams remain hopeful.
“This technology offers significant potential for advancing the understanding and protection of endangered species,” says Devine. “It will help CSIRO drive efforts towards ecological restoration.”