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,
https://imgur.com/a/m5vE3JT). 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 G
ononemertes 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
https://imgur.com/a/m5vE3JT). 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.