Biological rhythms in the beach amphipod Talitrus saltator

Lead Research Organisation: Aberystwyth University
Department Name: IBERS


All living things, from bacteria to humans have biological 'clocks' that enable them to keep time with their surrounding environment which is how we humans tend to wake up at roughly the same time each day! Remarkably, these internal timing mechanisms, which are normally found in the brains of animals, continue to work even when the organism is separated from cues, such as cycles of night and day. Biological clocks are extremely important because organisms can prepare in advance for bouts of activity, growth, mating and other necessary activity at the most appropriate part of the day to avoid predators, find food or mates. Therefore they contribute significantly to the survival and success of all organisms. We now know that the clock mechanisms in different plants and animals that have been studied share striking similarities in the molecules that drive them but, most of our understanding comes from work on a very few 'model organisms' such as the fruit fly. The fruit fly, and nearly all terrestrial organisms, uses the night and day light cycles to synchronise their clocks to local conditions. Consequently their rhythmic behaviour and physiology recurs on a cycle of about 24h. However, marine plants and animals are exposed to many different cyclic events such as the tide coming in and out (tidal clocks; every 12.4h), lunidian (cycles of tidal height; 12.8h) and even changes in tidal range (spring and neap tides) caused by the gravitational pull of the moon and the sun (semilunar clocks; 14d). Although it is well known that many marine species time their behaviour and physiology to these different events, we know very little about their how their internal clocks work. For instance, do they have the same molecular 'cogs' as daily clocks but 'run' at a different speed, or do they have dedicated 'tidal', 'lunidian' or 'semilunar' clocks? This project will decipher the genetic and biochemical basis of cyclic behaviours in the sand-hopper, Talitrus saltator. In the UK this small crustacean emerges from its burrow in the sand at night to feed. It navigates up and down the shore by moving towards the light of the sea horizon in the night but just before dawn it turns back and is drawn to the dark of the land. The emergence and light/dark preferences of the sand-hopper are under clock control and continue to be expressed even if they are kept artificially in total darkness. In the Mediterranean the same species navigate in a different way- by using the position of the sun and moon as their guide. This must also require a sense of time because as the Earth rotates, the sun and the moon appear to move across the sky and the sand-hopper must compensate for this to keep on course. I will compare the UK sand-hoppers to those of the Mediterranean to determine whether their clocks are geared differently or even if they have multiple clocks in their brains. The outcomes of this work could have important impacts on our understanding of biological clocks, how they have evolved and adapted to suit the prevailing environmental conditions and how they contribute to the success of the organism and their interactions with other species.

Planned Impact

The proposed project is in the realms of pure science. Nevertheless, the impacts of this work are tangible and real. Beneficiaries of the proposed research include:

1) Basic research groups across multiple biological disciplines- such as biomedical research, pure ecology, evolutionary and population biology. The importance of a more detailed knowledge of biological clocks should not be underestimated. For example, mutations in mammalian clock genes are directly linked to diseases including cancer, obesity, mental illness and sleep disorders. Given the level of conservation of clock molecules across all taxa, findings of the proposed work may have wider implications; defining novel systems and molecules in a non-model animal could, conceivably, transfer into more advanced systems. The project will produce a large volume of genetic data which will be publicly available. These data will be mined by workers across multiple scientific disciplines. In addition, antisera, which are routinely used in basic and applied research, will be produced and made available to bone fide researchers upon direct request to the proposer.
2) Aquaculture organisations and applied research and development in aquaculture. Most, if not all physiological processes are influenced by biological clocks. In arthropods this extends to moulting, reproduction and metabolism etc. Inappropriate or altered light and dark regimes are known to have deleterious effects on marine organisms with implications for commercially cultured species. However, light regimes are only one clock-entraining variable that coastal species are subject to. The proposed work will define clocks that operate with different periods- e.g. 24h, 12.4h, 24.8h etc. This information may be crucial in future attempts to assess the influence of changing environmental parameters on the welfare of commercial species both in captivity and in nature where anthropogenic interference via coastal engineering and lighting will impinge on the fauna and flora. The proposed work will also complement observations reported on the behaviour of wild, commercially harvested species (e.g. the scampi, Nephrops norvegicus). Analysis on the behaviour of this and other marine species has shaped harvesting strategies and catch data interpretation. Providing fine-scale insight into how their clocks function and respond to environmental change may indirectly influence our approach to marine harvest.
3) General Public. The mere concept of biological clocks is inherently fascinating to most people and has been the subject of popular science broadcasts on several occasions (BBC Horizon). Personal experience has taught me that the public are absorbed by the fact that even the most diminutive organisms exhibit an exquisite sense of time that can be used to organise behaviour such as orientation and navigation. Moreover, many apparently have no conception of how various (even common) animals behave and integrate within the ecosystem. Therefore, informing the public about the behaviour of organisms can add to quality of life and potentially even alter behaviour towards animals and each other. One of the first questions I am asked by members of the public when I talk about 'tidal clocks' or 'circadian rhythms' is "how do they do that?" It is therefore imperative that we have the necessary knowledge on these systems to give informed and understandable explanations. The proposed work will provide such detail.
Description This grant was designed to answer fundamental questions on the cellular and molecular control of rhythmic activity and celestial (moon and sun) compass navigation in a small beach crustacean; as such it was largely in the realms of pure science.
The experimental subject, the sand-hopper, Talitrus saltator lives buried in sand at the strand-line on the top of sandy beaches and at night emerges to forage and reproduce. In Talitrus the timing of this nocturnal activity is governed by an internal clock mechanism so that it is not exposed to increased risk of predation and desiccation during daytime. Sand-hoppers also use a the sun (if disturbed from the sand during daytime) and moon (when foraging) as compass guides to navigate appropriately up and down shore. However, this means they must continuously adjust their bearing relative to these celestial cues to compensate their apparent movement over time (due to Earth's rotation). Competence to make these compensatory movements necessitates a sense of time (time-compensated orientation). At the time of writing the grant, nothing was known about the molecular underpinnings of the clock mechanism(s) that govern a) rhythmic activity and b) time-compensated orientation. Thus, the project objectives were:

I. To isolate and characterize putative circadian clock genes from Talitrus and quantify their expression by qRT-PCR
II. To perform high-resolution, transcriptome-wide quantitative analysis of rhythmic gene expression in behaviorally rhythmic Talitrus by Illumina 'RNAseq' over a 24h period.
III. To generate antisera against core clock components for anatomical mapping of the pacemaker cells in Talitrus.

The grant was active for 24 months during which time the principle project aims were met, and indeed exceeded. Significant outcomes from the work are detailed below:

1. Measurement of activity patterns in Talitrus was pivotal to the thorough examination of its clock-controlled behavior. I designed a bespoke infrared recording device in consultation with TriKinetics Ltd (Waltham, MA) that yielded excellent data, enabling real-time monitoring of multiple individuals over many days. These rigs were used throughout the grant period (and continue to be used) and enable us to monitor behaviour in response to altered environments (such as light regimes).

2. We generated a comprehensive suite of so-called clock gene sequences expressed in Talitrus brains by homology cloning and RNAseq and defined their expression patterns in animals allowed to 'free-run' in constant conditions over a 24h period. Thus, we have defined genes that are likely involved in endogenous clock function and represent suitable targets for future manipulation (e.g. by dsRNAi and genome editing-CRISPR) and assessment of their functional role. For instance, the canonical clock genes period, cryptochrome2 and timeless show cycles in abundance over a 24h time-course. These gene expression profiles share similarities in gene cycling with other key invertebrate models including monarch butterflies, which like Talitrus, use time-compensated sun compass navigation and offer us a valuable evolutionary perspective on the clock mechanisms.

3. Our high quality database of all genes expressed in the heads of Talitrus throughout a day-night cycle using new 'Next Generation Sequencing (NGS) strategies has been deposited in a public database for the benefit of the scientific community (see XXX field of Research Fish).

4. We formed a new collaboration with a group in Italy (Ugolini group, Firenze) who are expert in monitoring sandhopper orientation and demonstrated that clock gene activity can be phase shifted by altering the light/dark cycles under which the animals are kept. This shifted molecular clock mechanism is mirrored with a corresponding deflection in orientation angle in their orientation behaviour. Thus we believe we have shown the molecular correlates of circadian timing and time-compensated orientation behavior.

5. We showed that the antennae of Talitrus house a timing mechanisms that is integral to lunar orientation but not to solar orientation. At the same time, we provided evidence that the antennae express cycling transcripts for the clock genes period and cry2 but with cry2 in anti-phase to that in the brain. These results provide tantalizing insights into the putative lunar clock that is emerging as an important mechanism in a range of marine (but also terrestrial) species. Work is now ongoing to fully characterize each system (solar clocks and lunar clock) by attempting to localize and visualize the cells of each system in the brain and antennae. To do this we have generated an antiserum that recognizes epitopes in the PERIOD protein (the transcripts of which show robust cycling in free-runing individuals). Used in fluorescent immunohistochemical preparations of whole brains, this serum has revealed at least 4 putative clock cells in the protocerebrum (front part of the brain) of Talitrus as for other arthropod clock cells (see Figure 1). Work is ongoing to validate and extend these findings (PhD stipend to Laura Hoelters) and to establish staining patterns over a 24h interval. These data will shed light on the cellular dynamics of Period expression in the brain. At the same time we will explore the antennal clocks with this antiserum and with a newly acquired CRY2 sera (purchased with funds awarded via the Aberystwyth University Research Fund).

Figure 1. Anti-TalPER immunoreactivity in the brain (protocerebrum) of T. saltator. Left image shows nuclear and cytoplasmic staining as well as axon labeling, whilst the right image shows no axonal staining. Scale bars depict 20µm.

Looking forward, I am currently in the planning stages of writing a grant to develop CRISPR in Talitrus using the genetic data I have described here. To strengthen my case, I will soon (July 2017) be travelling to UC Berkeley to the Nipam Patel Laboratory (where genome editing is done routinely) to learn the protocols needed for this exciting approach. We anticipate that by generating clock gene knockouts we will unravel the molecular clockwork that underlies the clock -driven behavioural phenotypes we have described. The clear and robust behavioural phenotypes of Talitrus make it particularly amenable to monitoring the effects of gene knockout. Development of CRISPR in Talitrus will also elevate it towards becoming a model crustacean, albeit requiring a fully annotated genome!
Furthermore, I have been invited to the laboratory of Bambos Kyriacou (Leicester, UK) to use a Drosophila S2 cell luciferase reporter assay to define the interactions and putative roles of Talitrus clock proteins (Period, Cry 2, Bmal, Timeless and Clock) in the core oscillator of this animal- whilst clock genes and proteins are highly conserved there is some degree of functional redundancy. Establishing the interactive roles of the core clock elements in Talitrus, as we have done in the closely related isopod, Eurydice Pulchra (Zhang et al, 2013, Current Biology) we will begin to define the clock mechanism in crustaceans and how they differ from more evolved species (crustacean pre-date flies by ~400 million years!).
These research visits have been financially supported by my University via the Aberystwyth University Research Fund and by awarding me research. The grant application, backed by the outcomes of these studies together with outputs from the NI grant, will be written in collaboration with Prof Ugolini in Firenze, thus strengthening our European collaboration.
Exploitation Route Our sera, Following validation, will be made available to the scientific community; antisera for clock proteins in non-model systems are rare and ours will will expand the current inventory of these. There is growing attention being paid to rhythmic biology of crustaceans such as krill, crabs and copepods but, at present, suitable resources for cellular localization are limited. As alluded to above, I am continuing to develop antisera for clock proteins in crustaceans that will provide a valuable resource for the clock community.
Our transcriptome data is publicly available and be of benefit to arthropod biologists world-wide as a source of sequence information on a crustacean system. Indeed I have been approached by the Decapod Crustacean Transcriptome (DCT) Research Colloboration Network (hosted in the US), that aims to create an international network for crustacean biologists with access to a secure wiki space for asking questions, sharing solutions and discussing results in pursuit of various Decpod RNA-Seq studies.
Sectors Other

Description The original proposal for this work was explicit in its declaration that the work was in the realms of pure science and it would be disingenuous to pretend we have made massive contributions to non-academic beneficiaries. However, during the course of this work we have generated data that may benefit aquaculturists; we have demonstrated the molecular and behavioural influence of altered light regime on intertidal animals. Our reagents and databases are a valuable resource to the scientific community and these will be freely available by request (antibodies) and via public databases (genetic information). Our findings have been reported to the lay audience via BBC television interview (2015 viewing, BBC2 Coast) and by presentation at Science Cafe (November 2014), public events such as the Aberaeron Seafood Festival (2013 and 2014) and through the Aberystwyth School of Education and Lifelong Learning. In addition our work has been used to engage school children via the Gene Park Wales, Genetic Roadshow (2013, 2015 and 2016) and National Science and Engineering week (all years from 2013).
First Year Of Impact 2013
Sector Education
Impact Types Cultural,Societal

Description Aberystwyth University Doctoral Career Development Scheme
Amount £43,500 (GBP)
Organisation University of Wales 
Department Institute of Biological Environmental and Rural Sciences (IBERS)
Sector Academic/University
Country United Kingdom
Start 10/2013 
End 09/2016
Description Capital Equipment Bid (BBSRC Year End funds)
Amount £130,000 (GBP)
Organisation University of Wales 
Department Institute of Biological Environmental and Rural Sciences (IBERS)
Sector Academic/University
Country United Kingdom
Start 04/2014 
End 03/2015
Title Anti-Talitrus Period serum 
Description We have raised an antiserum against the Period protein (a peptide fragment of) from Talitrus saltator and made in rabbit. This is under characterisation at present. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact There are currently very few non-model species antisera against core circadian clock proteins. Our Period protein will become available to other researchers in the scientific community for the localisation and, subject to validation, quantification of clock proteins. 
Title Talitrus saltator brain neurotranscriptome 
Description We have generated a high quality neurotranscriptome from Illumina HiSeq processing of RNA taken across a 24h temporal window to capture genes expressed at different times. We assembled the reads from Illumina sequencing using two packages, Velvet and Trinity and annotated the Trinity assembly using BLAST2GO. All data will be made available via the NCBI repository once we have submitted a key manuscript from this work (early 2015). 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact We published this database in 2016. It is anticipated that this rich genetic resource will inform other workers in arthropod biology. 
Description Collaboration with Firenze University, Italy 
Organisation University of Florence
Country Italy 
Sector Academic/University 
PI Contribution The NERC NI funding has resulted in a new and exciting collaboration with Firenze University (Professor Alberto Ugolini). Professor Ugolini is an expert on the navigational behaviour of Talitrus saltator and has significantly improved and streamlined our tissue sampling and experimental work in this area (Programme of work 2, phase II). This work is ongoing and during the summer of 2014 we will undertake a major sampling effort in Italy with Professor Ugolini.
Collaborator Contribution Professor Ugolini has helped us conduct behavioural experiments on sand-hopper orientation and the effect of altered photic regime on orientation angle. We have now compared these behavioural outputs, taken with Professor Ugolini, with the expression of key circadian clock genes that we think underpin the time-compensation of solar navigation in these animals.
Impact The work with have conducted in collaboration with Professor Ugolini is currently being prepared for publication. Data collection and analysis is complete and manuscripts are being finalised.
Start Year 2013
Description Aberystywth Science Cafe (November 2014) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This session is due to take place on 17th November 2014; still within the active life of the grant. The Science Cafe is, regionally, extremely popular in sharing knowledge and scientific endeavour with the lay public. I will be running live demonstrations of Talitrus saltator's behaviour and engaging in a Q&A session with the public on my NERC funded research.

The event is due to take place on 17th November
Year(s) Of Engagement Activity 2014
Description BBC2 Coast television programme 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact In September 2014 I was invited by the BBC to film a short piece for BBC2s Coast programme. During filming I demonstrated the organism my research focusses on (Talitrus saltator), its behavioural biology and wider importance to shore ecology. The filming is now being edited for screening in 2015.

This filming has not yet been screened but resulted from the BBC researcher viewing my earlier contribution to BBC4's "Secret Life of Rockpools" science documentary that received over 1.5m viewers.
Year(s) Of Engagement Activity 2014
Description GenePark Wales, Genetic Roadshow event 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact "During the autumn 2013 and spring 2014 terms, over 4000 students from 73 schools and colleges across Wales and the border counties heard talks from 58 speakers from research, healthcare and the police force on the latest advances in genetics and genomics and about genetics-related courses and careers" Taken from Wales GenePark website to summarise the objectives and impact of these educational events. Website URL is given below. My talk was on biological clocks to over 200 students from around Wales. My talk was very well received and managed to relate my basic science on marine organisms beyond my own discipline to ecology, agriculture and human health.

Feedback was gathered from students and teachers after the event and analysed by GPW. The data are summarised: Scores on a Likert scale where 1= disagree strongly, 5= agree strongly
Teachers (mean score):
Overall content- 5
Relevance to syllabus-2
Educational value-5

Students (mean score):
I enjoyed listening to this talk- 4.9
I learned new things about this topic- 4.9
I am more aware about a career in this field- 4.3
Year(s) Of Engagement Activity 2013