Parasites alter interaction patterns in fish social networks

Social networks influence the spread of parasites through populations. Although we know how parasites are transmitted as a product of social interactions, we have a limited understanding of how social networks are affected by parasites over time. Host–parasite interactions and the networks they form, are typically examined as static networks, and while topological descriptions at a specific time point are useful, both behaviour and the infection process are dynamic. By monitoring replicate populations of Trinidadian guppies (Poecilia reticulata) daily before and during infection with the ectoparasite Gyrodactylus turnbulli, we show how parasitism drives social network dynamics. Specifically, infected individuals increased their connections in networks affected by parasitism. In contrast, uninfected control shoals showed no change in network metrics. The structure of subnetworks (motifs) and networks, however, did not change in response to infection status. These findings provide further evidence of reciprocal host behaviour–parasite feedback mechanisms, and highlight that infected fish alter their interactions in order to ‘off-load’ their parasites. Understanding how these reciprocal interactions affect the structure and function of natural systems, as well as understanding how these interactions may alter with future environmental change, are key areas of future research.

Methods
Host-parasite system

Trinidadian guppies (Poecilia reticulata) were laboratory-reared descendants of wild-caught stock from the Lower Aripo River, Trinidad, in 2012. Fish were initially housed at the University of Exeter, before transfer to Cardiff University in 2014 to be maintained in 70 L dechlorinated water tanks under standard conditions of 24 ± 0.5°C on a 12 h light: 12 h dark photoperiod (Lights on 07:00–19:00). Fish were fed daily on Aquarian® Tropical fish flakes, subsidised with freshly hatched Artemia salina and adult Daphnia magna. Aquaria were checked weekly for fry, which were transferred to rearing tanks from which female fish were isolated at 8-12 weeks. Only female guppies (n=120) were used due to their greater propensity to shoal than males (Griffiths & Magurran, 1998), but also to avoid the confounding effects of male courtship behaviour and sexual interactions on parasite transmission and social network structure.

For experimental infections, we used the isogenic Gt3 strain of Gyrodactylus turnbulli, which originated from a single worm isolated from an ornamental guppy in 1997. This ectoparasite population has since been maintained in culture as described by Stewart et al. (2017). The monogenean worm is a common ectoparasite of guppies in both wild and ornamental stocks, and has a range of physiological and behavioural impacts (Croft et al., 2011; Stewart et al., 2017; Arapi et al., 2024). It is directly transmitted, transferring from host to host when the fish contact one another and has a short generation time, giving birth to live (already pregnant) young that attach to the fish alongside the parent worm (Bakke et al., 2007). To experimentally infect a fish, an infected (donor) fish from the culture was sacrificed via cranial destruction, and the caudal fin brought into close contact with a naïve (recipient) guppy, which had been temporarily anesthetised with 0.02% tricaine methansulfonate (MS-222). The transfer of parasites was observed under a dissecting microscope with fibre-optic illumination, following the standard methods of King & Cable (2007). Control fish (i.e., sham infected) were handled and exposed to anaesthetic in the same manner as the experimental fish but without exposure to parasitic infection. Parasite infections were monitored non-destructively throughout the experiment by again briefly anaesthetising each fish in a shoal (including the control shoals) and counting the number of external worms on the surface of the fish using a dissecting microscope.

Experimental setup

Experimental trials took place in a 70 L tank of dechlorinated water, maintained under standard light and temperature conditions (see Host-parasite system). A 2 cm layer of fine gravel substrate filled the base of the aquarium, which was lit from above using daylight mimicking strip lights (Sylvania T5 F13W/54-765 G5 Luxline Standard Daylight bulb) diffused by white fabric. The chamber was surrounded on three sides with opaque white fabric to prevent external disturbances, with one side left open to allow for observations.

Behavioural experiments

A total of 20 replicate shoals, each containing six sized-matched female P. reticulata, were monitored daily for 10 days, with experimental infection occurring on day 5 in 15 randomly selected replicates, and a sham infection in the remaining five controls. Each fish was uniquely marked using visual implant elastomer (VIE), enabling individual fish identification during a trial. To do this, fish were briefly anaesthetised using 0.02% MS222, and VIE injected into the ventral or dorsal muscle tissue. This is a marking procedure extensively used in guppies (Croft et al., 2006, 2009; Hasenjager & Dugatkin, 2017; Wilson et al., 2015) that does not appear to influence social behaviour (Croft et al., 2004).

Fish standard length (SL; mm) was measured before each group was placed into a separate 5 L aquarium to form shoals over a 2-week familiarisation period (Griffiths & Magurran, 1997), before transferring to an experimental chamber to acclimate for 24 h.

On day 5, all fish were temporarily isolated in individual 1 L pots and either the most or least connected shoal member (determined by assessing accumulated contact frequency data until day 5; see ‘social network construction’, below) was infected with exactly 30 G. turnbulli individuals. This procedure formed three experimental treatments: most connected infected (n = 7 shoals), least connected infected (n= 8 shoals), and uninfected controls (n = 5 shoals). The unbalanced experimental design arose through limited availability of mature female fish for the experiment. Despite uneven sample sizes, an adequate number of replicates ensured that robust statistical analysis comparing experimental treatments could be performed. Within each infected shoal, a single fish was experimentally infected and the remaining five fish in each shoal, as well as each fish in the control groups, were sham infected by anaesthetising and manipulating under the microscope, but without exposure to parasites. Fish were revived in 1 L dechlorinated water and returned to their shoal groups. Infection was confirmed on day 6, and each fish was screened on consecutive days thereafter (days 7, 8, 9 and 10) to quantify G. turnbulli intensity, following behavioural observations. At each time step control and experimental groups underwent the same experimental procedures; anaesthesia followed by handling.

Social network construction

For each shoal, interactions were monitored on each day (1-10) for a 10 min period (between 9:00–12:00, 3 shoals per experiment). The frequency of interactions between individuals (the number of direct contact events; e.g., skin-skin contact including a bite or the brushing of fins, typically lasting <1 sec) was recorded for all individuals, as well at the directionality of the interactions (i.e., which fish initiated the interaction). This resulted in a series of directed, weighted networks, where individuals are represented by nodes and interactions between individual fish by edges. Each behaviour recorded was directional such that we could record who approached whom giving us the ability to quantify the number of outgoing contacts from shoal mates (‘out degree’) and the number of incoming contacts (‘in degree’). Because multiple interactions can occur over time these edges, or interactions within the network were weighted; i.e., were a simple count of how many times they occurred.