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Student Project

Endosymbionts of Pyura praeputialis near Brisbane, Australia

Maximiliaan Koebrugge 2020


Pyura praeputialis is an important ecosystem engineer, providing suitable habitat for, and influencing the distribution and abundance of an array of other species. However, little research on its endosymbionts has been conducted. This study aimed to determine the endosymbionts of Pyura praeputialis from two different locations on the intertidal rocky shore of South-East Queensland. The commensal copepod, Doropygus pulex, has been associated with the branchial sac of P. praeputialis and the number of copepods per ascidian seemed to increase with size. No difference in copepod abundance has been found between the sites, which reflects its cosmopolitan distribution. Female D. pulex were found more frequently than males, with a sex ratio of 1:0.09, possible indicating a new behavioural trait for this species. Besides copepods, observations of three unidentified species have been made, including a slender worm in the anus and three white specimens and a amphipod in the branchial sac. However, the organisms occurred in low numbers and remained unidentified, therefore the evidence for possibly novel endosymbionts remained inconclusive. Future research should aim to fill in these gaps by identifying the species of the unidentified observed organisms and their possible endosymbiotic relationship with P. praeputialis.


Dominant species are often described as ecosystem engineers, as they provide suitable habitat for, and influence the distribution and abundance of an array of other species (Rius, Teske et al. 2017). Species of the Pyura stolonifera species complex (Rius and Teske 2011) are ecosystem engineers of considerable ecological importance in rocky shore communities on the southern hemisphere, since they strongly influence the local community (Rius, Teske et al. 2017). Species that are dominant in their native habitat have a higher change of becoming an invasive species once it is established elsewhere (Simberloff 2010). Since, Pyura species are a dominant species in intertidal and subtidal areas in their native range, they have the potential to become invasive once established elsewhere. When that happens, their abilities as ecosystem engineer enables them with the potential to strongly alter local communities. Pyura praeputialis is reported to be invasive in the bay of Antofagasta, on the coast of Chile, where it has replaced the native mussel (Castilla, Manriquez et al. 2014). P. praeputialis, is a keystone species as it shapes the ecosystem, determining the entire species composition. Therefore, fundamental research on the biology of these ecosystem engineers is important. 
Research on endosymbionts of Pyura stolonifera has been performed before (Day 1974, Oldewage 1994, Dalby 1996), however, no research has been conducted on endosymbionts of Pyura praeputialis from the Australian East coast. Notwithstanding its status as a dominant ecosystem engineer in the area, shaping the distribution and abundance of many other organisms (Monteiro, Chapman et al. 2002). Little is known about the biology of endosymbionts, as they are cryptic; restricted to life in their host, making it a hard study subject. 
This study aimed to determine the endosymbionts of Pyura praeputialis from two locations in South-East Queensland. The findings will be compared with previous studies, in particular with the study conducted by Dalby (1996), as he conducted a similar study 24 years ago in Southern Australia. It is expected that the results from this study will reflect previous findings by Dalby and others. 
The two different locations in South-East Queensland are almost 200 km apart and habitat and environmental conditions differ between the two. The southern site is close to Coolangatta, were ascidian were exposed to large wave energy and long periods of emergence. The northern site is close to Yaroomba, were ascidians inhabited intertidal pools (that had a water flow), which were more protected from higher energy wave impacts. The northern site is located in the zone where temperate and tropical water mix, which could influence the endosymbiotic community.

Materials and Methods

Specimen collection

Pyura praeputialis (Heller, 1878) were collected from the intertidal rocky shore of Queensland, Australia at two sites: Kirra beach and Point Arkwright, in May 2020 (Fig. 1 and 2). A total of 31 specimens was collected, 15 from Kirra beach and 16 from Point Arkwright. P. praeputialis were removed from the rocky substrate using a chisel and a hammer after methods by Dalby (1996). After removal, the ascidians were kept in containers with seawater until dissection (within 36 h). The length, from test to buccal siphon, and the diameter, from the top where it is widest, were recorded prior to dissection. For every ascidian the volume (length x diameter), cavity size, symbionts, corresponding location and gonad index were measured. 

Figure 1
Figure 2


Dissection was necessary to record all internal symbionts. The following approach was followed for all specimens. The ascidian was cut open along the mid-sagittal axis, through both siphons (Fig. 3). The body was removed from the tunic and symbionts inside the tunic, branchial sac and anus were recorded. The branchial sac was cut out and washed in filtered seawater to ensure all symbionts were recorded (Fig. 4). The far end of the intestine and the anus were checked by washing samples of digestive material. Examinations were done strictly with the naked eye, so small symbionts were not seen. After the body was removed from the tunic, the inside cavity was documented by measuring the inside length and diameter at its longest point. Copepods, that were discovered in the ascidians from Point Arkwright, have been identified to species level by using a microscope in one of the labs at the University of Queensland.
Figure 3
Figure 4

Gonad Index

Ascidian gonad maturity was measured using an adapted version of the gonad index (GI) (Dalby 1996), ranging from 0 to 3. Gonad index was measured relative to the body size, where 0 = gonad absent or small, with no gametes visible, 1 = gonad small to medium sized, still undeveloped, with no gametes visible, 2 = gonad developed, medium sized, with some gametes visible and 3 = gonad large and swollen with gametes (white, milky sperm and dark greenish eggs). 

Copepod displacement

To examine how symbionts invade their host, 5 copepods that were collected from a dissected specimen of Point Arkwright were placed in a container with a live filterfeeding P. praeputialis for one hour. Their movements during, and presence after the hour were noted. 


Statistical analysis was performed in RStudio, using R software, version 1.1.463. Mann-Whitney U tests were conducted to compare the difference in copepod abundance between locations and the difference in the average number of females and males per ascidian. The three different size measurements, volume, cavity area and GI were compared using a Pearson correlation test. The relationship between copepod abundance and size was analysed in two ways. To test for differences in copepod abundance per GI, an Anova was conducted between the number of copepods and GI. The relationship between copepod abundance and size was examined using a linear regression model. 


P. praeputialis from both locations were occupied by symbiotic copepods, Doropygus pulex (Thorell, 1859) (Fig. 5). Overall prevalence (percentage of hosts occupied) by D. pulex was 61% and intensities (number of copepods per individual host) ranged from 1 to 9 copepods. The total number of copepods was higher at Kirra beach (35) compared to Point Arkwright (15). However, no significant difference in average abundance of copepods was found between ascidians from Kirra beach (2.33 ± 0.60) and Point Arkwright (1.50 ± 0.55).
      Copepods only occupied the pharynx, particularly the branchial folds. Gross health of occupied and vacant hosts did not seem to differ, D. pulex did not appear to damage the pharynx; no cysts were found in any occupied body parts.
  Both adult females and adult males were found to occupy the host, however females were far more frequent than males, with a sex ratio of 1:0.09. The mean number of females per host (1.77 ± 0.39) was significantly (p = 5.978e-06 ) higher than the mean number of males (0.13 ± 0.07).
  Three measurements of size were taken for all ascidians: volume, cavity area and GI. The validity for each as a measurement for size was tested, which yielded that all factors were strongly correlated to one another: volume and cavity area (r = 0.93), GI and cavity area (r = 0.88) and GI and volume (r = 0.86). 
  There is a positive trend between copepod abundance and Gonad Index (Fig. 6). Ascidians with a GI of 1 (2.00 ± 0.577), 2 (2.57 ± 0.972) and 3 (5.25 ± 1.31) have a significantly (p = 0.18; 0.18 and 0.10, respectively) higher copepod abundance than ascidians with a GI of 0 (0.462 ± 0.215). No difference in copepod ascidians has been found between the other groups of ascidians. 

The observed trend is best illustrated by a linear regression between copepod abundance and cavity area, this is justified by the close relationship between cavity area and GI (r = 0.88) (Fig. 7). The linear regression model shows a significant relationship between copepod abundance and size (p = 0.0001), however it has to be analysed with caution, due to of the poor fit with the observed data (R2 = 0.38).

Besides these quantitative results, some qualitative discoveries have been made as well. From the end of the digestive tract, the anus, of one of the ascidians from Kirra beach, an unidentified worm, most likely a nemertea, has been found (video 1, 
Three small unidentified white specimens have been found in the branchial sac of P. praeputialis from Point Arkwright, as well as one individual of unidentified amphipod (video 2 and 3, 

All copepods collected from the infauna were highly immobile, only able to move in a wriggling motion by squirming their urosome up and down. When they were placed in a container with a filter feeding ascidian this behaviour did not change, after one hour all copepods were still at the bottom of the container. 

Figure 5
Figure 6
Figure 7


Doropygus pulex was site-specific in Pyura praeputialis: D. pulex was only recorded in the pharynx. Other research on D. pulex and other ascidicolous copepod species supports this finding, as many of the species are found to only occupy the branchial sac (Gotto 1979, Dalby 1996). The copepods occupy the branchial sac, so they can take advantage of the filter feeding performed by their host. Gotto (1979) refers to the feeding style of copepod species living in the pharynx as ‘larder feeders’: commensal copepods that remove food particles from the mucus sheets that line the pharyngeal wall. 
Copepods were more often found in large than small hosts and copepod abundance seems to increase with size. This trend could be caused by the fact that invasive copepodites encounter large ascidians more often, either because large ascidians filter more water than smaller ones or, simply, because they are larger targets. Another explanation could be that the invasive copepodites select large ascidians as they offer a superior habitat; more available space and a higher water flow, resulting in a higher food availability. However, the superior habitat in larger ascidians could also result in a higher survival of copepods, resulting in a similar abundancy trend. Invasive copepodites of other species depend on chemotactic cues for host selection (Gotto 1979) and invasive copepodites of other Doropygus species have evident sensory systems (Dudley 1972). Therefore, it is possible that copepodites of D. pulex could actively select preferred hosts. 
In addition, large ascidians have been around for a longer time than smaller ascidians and have, therefore, been available for recruitment for a longer time. However, the ‘age-hypothesis’ is unlikely in the case of copepods, because of their short lifespan. D. pulex in Sweden reach an age of approximately 15 months and copepodites disperse after hatching (Svavarsson, Svane et al. 1993). 

No difference in copepod presence or abundance was found between the different sites. This corresponds to its distribution range, as D. pulex is a cosmopolitan species. D. pulex has been found in the North Sea (Svavarsson, Svane et al. 1993), the Mediterranean (Pastore 2001), Western Atlantic (Wilson 1932), South-Africa (Day 1974), Southern Australia (Dalby 1996), and Eastern Australia. The wide distribution over a variety of different habitats, clearly shows that D. pulex is able to survive in a broad range of environmental conditions. The two different habitats sampled in this study do not differ as much from each other as the ones mentioned above, therefore a difference in presence or abundance of D. pulex would not be expected. 

Gross health between occupied and vacant hosts did not seem to differ, therefore Doropygus pulex is suspected to be commensal in Pyura praeputialis. Dalby (1996), who investigated symbionts in extensively more specimens, found up to 158 copepods in a one individual P. praeputialis. He concluded that if one individual can host up to 158 copepods without apparent effects on the gross health the relationship must be commensal. 

This study found a significantly higher female abundance, with a sex ratio of 1:0.09. This is inconsistent with previous findings, as frequency for female and male Doropygus pulex has been reported to be equal, year-round (Svavarsson, Svane et al. 1993). Males are orange-coloured, as is the branchial sac, making them well camouflaged (authors personal observations). Therefore, it is possible that a lot of males were not recorded during dissection. However, it seems unlikely, as branchial sacs were washed to ensure all symbionts were recorded. Moreover, such a large discrepancy cannot be solely explained by unrecorded individuals. Males from other symbiotic copepod species have been hypothesised to show intermittent absence in response to lower food supply, to reduce intraspecific competition (Svavarsson, Svane et al. 1993). Male presence is not required at all times, because female copepods are able to store sperm in spermatophores. It is possible that the Doropygus pulex species observed in this study has diverged from the D. pulex observed by Svavarsson et al., and, therefore, exhibit a different frequency. 
Furthermore, dimorphism is common in copepods, with males reduced to dwarf-males in some species (Gotto 1979). Dudley (1966) found male dimorphism in Doropygus seclusus. Copepodites moulted either into anamorphic or metamorphic adults, behaviourally distinct ‘active walkers’ or ‘efficient swimmers’ (Gotto 1979). Male dimorphism may be present in the Doropygus pulex species observed in this study, as D. pulex is a cryptic species and much about its lifecycle remains unknown. Male dimorphism could perhaps explain the discrepancy between the observed frequencies. However, this seems improbable and more studies on the lifecycle of D. pulex have to be performed before such hypotheses could be confirmed. Therefore, it remains uncertain what causes the difference in female and male frequency of D. pulex found in this study.

One Pyura praeputialis from Kirra beach hosted an unidentified slender worm in the end of its digestive tract (video 1, The species could not be determined, as access to proper equipment was limited. It possibly is a nemertean, order hoplonemertea, as these exhibit commensalistic and parasitic feeding modes (Gibson 1974). No symbiotic nemerteans living in Pyura praeputialis have been reported before. There have been very few confirmed reports of nemerteans living in symbiosis with ascidians (Dalby 1996), making this observation a rare finding. Dalby found Gononemertes australiensis inhabiting the “yellow morph” of Pyura stolonifera, now known to be Pyura dalbyi (Rius and Teske 2011). Even though he also investigated endosymbionts of the “brown morph”, Pyura praeputialis, he reports that D. pulex and G. australiensis are host-specific. The present observation of a worm in the anus of P. praeputialis could indicate that there are other species of nemerteans occupying Pyura species. However, only one unidentified worm was found, which makes the possibility that this is an endosymbiont not very strong, as it could be a sporadic finding or a contamination from the epifauna.
On top of that, three small unidentified white specimens and one unidentified amphipod were found in P. praeputialis from Point Arkwright (video 2 and 3 Dalby (1996) found amphipods, Paraleucothoe novaehollandiae, in both Pyura species, however, limited exploratory determinations conclude that the current amphipod observation is not the same species as found by Dalby. It possibly is a contamination from the epifauna, but it may be a novel endosymbiont, as the environment at Point Arkwright is different than where Dalby performed his research. Nonetheless, one unidentified observation is not strong enough to propose such a hypothesis, hence more research is needed.
In addition, three unidentified white specimens were found in Pyura praeputialis from Point Arkwright. Possible options are that they are (1) juvenile copepodites, (2) a dimorphic male, or (3) a new species of endosymbiont. (1) The three unidentified white specimens could be juvenile copepodites. They are not larvae (nauplii), comparison to nauplii of Doropygus porcicaude indicated that nauplii look morphologically very different from the specimens observed (Gray 1933). It is hypothesised that the white specimens seen, could be copepodites of D. pulex and perhaps were in the dispersive stage, as they seemed (observations with the naked eye) similar to small male specimens, elongated body and they were relatively mobile. It was suspected to be a juvenile due to the apparent white colour and high mobility. Images of copepodites of Doropygus species or Notodelphids are not readily available, so no conclusive evidence can be given. (2) Some copepod species display male dimorphism, it is suggested that the white specimens observed could be dimorphic males, since they looked similar to male D. pulex, but smaller and white. However, this seems improbable, since the specimens were only reported in two ascidians from one location and no record of male dimorphism in D. pulex has been made before. (3) The unidentified white specimens could be a novel endosymbiont, however this cannot be confirmed as the specimens have not been examined in greater detail. If it were to be a novel symbiont, it could indicate a difference in endosymbiotic fauna between the two locations. 
It seems unlikely that the observation of the white specimens is caused by a contamination from the epifauna as three specimens have been found in two hosts. Moreover, two out of three specimens have been recovered when the branchial sac was washed with filtered seawater, indicating they were residing inside the ascidian. 

Copepods were highly immobile, only able to move in a wriggling motion by squirming their urosome up and down. This observation led to questions about their ability to invade a host, therefore 5 individuals were placed in a container with a filter feeding ascidian to investigate whether this changed their behaviour. It was found that their mobility did not change and they did not move from the bottom of the container, which indicates that Doropygus pulex must invade their host at an earlier stage in its lifecycle. 
Dudley (1966) investigated the life cycle of four Doropygus species and found that they pass through 5 nauplii stages, in a time span of 96-108 hours, followed by 5 copepodite stages before moulting into their sixth, adult form (Gotto 1979). Dudley found strong evidence that the second copepodite stage is the infective stage as a moulting into the third stage is only achieved within the host ascidian, in some species (Gotto 1979). Moreover, the eye becomes functional in the second copepodite stage of Doropygus seclusus, albeit smaller than that of the adult, its functionality is similar. Therefore, the host-infective stage is marked by negative phototaxis, with the copepod sinking down to invade its benthic host (Dudley 1969). Furthermore, a specialised chemosensory organ reaches its greatest development in the second copepodite stage of Doropygus seclusus (Dudley 1972). The organ is proportionately smaller in later copepod stages, therefore Dudley (1972) suggested that the organ is involved in host recognition during the invasive stage. 

This study conducted a similar study as Dalby (1996), in a different location. Substantial differences were found compared to the study conducted by Dalby. First of all, the intensities (number of copepods per ascidian) ranged from 1 to 9 in this study, versus 1 to 158 in the study by Dalby. The number of ascidians investigated is considerably lower in this study than in the study by Dalby, which could explain the difference. 
In addition, Dalby found amphipod, Paraleucothoe novaehollandiae, presence in 20 to 31% of Pyura praeputialis, while this study only reports one observation of an unidentified amphipod. Other amphipod species are reported to be absent in their host during spring/summer, allowing copepods to become abundant (Thiel 2000). When amphipods occupy their host again, copepod abundance decreases dramatically. It is possible that such a mechanism is active in the P. praeputialis examined and that amphipods were not abundant at the moment of collection. 

The copepod species has been determined to be Doropygus pulex, because of three distinct features. First of all, the urosome is always turned at a right angle to the metasome and sometimes even forward. This has been put forward as a sufficient to distinguish this species (Wilson 1932). However, new Doropygus species have been described since Wilson published his book, therefore more features were investigated. The fifth leg comprises of two segments, with the basal being wider three-fourths as long as the terminal segment and the terminal segment three times as long as wide. This makes it significantly different from new described species that look similar (Oldewage 1994). Another characteristic that is used to determine the species is the number of setae on the different segments of the ampullenes. Nevertheless, specimens were not in the best state, therefore the two features described above and findings by Dalby (1996), were assessed as sufficient to determine the copepods to be Doropygus pulex.

Studies performed by this cohort of BIOL3211 will be known as the ‘covid-affected’ studies, as social restriction measures greatly affected the available support and resources. Many of the results found in this study could not be properly investigated due to a lack of adequate resources. In addition, some valuable papers (Dudley 1966, Raibaut 1985, Trilles 2012) could not be accessed due to social restriction measures and the short time scale of putting together this article. Lastly, copepods were preserved in methylated spirits (95% ethanol, 5% methanol), which discoloured them, thereby impeding species determination even further. Future students aiming to investigate the external anatomy of copepods should preserve specimens in lactic acid (Humes and Gooding 1964).

In conclusion, this study confirmed the presence of Doropygus pulex in Pyura praeputialis on the intertidal rocky shore of South-East Queensland. The commensal copepod has only been associated with the branchial sac and the number of copepods per ascidian seemed to increase with size. No difference in copepod abundance was found between the different sites, reflecting its cosmopolitan distribution. Female D. pulex were found more frequently than males, with a sex ratio of 1:0.09, possible indicating a new behavioural trait for this species. Besides copepods, three possibly novel endosymbionts were observed, however, these organisms occurred in very low numbers and remained unidentified, providing no conclusive evidence. Future research should try to fill in the gaps left open by this study, by aiming to identify the species of the unidentified observed organisms and their possible endosymbiotic relationship with P. praeputialis. Furthermore, future research could focus on the cryptic life cycle of D. pulex.


First of all, I would like to express my gratitude to Robyn Davies, for help with specimen collection and dissection. I benefitted greatly from the Ph.D. work done by Dalby, so special gratitude is owed to him. Furthermore, I would like to thank Professor Sandie and Bernard Degnan for the valuable lessons on marine invertebrates. Moreover, I would like to express my gratitude to Dr. Gurion Ang for access to his lab to identify the copepods. Lastly, I would to express special gratitude to Emily Faye and Jarred Vardy for continous support, peerreviewing and technical support.


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