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Microhabitat preference of the Banggai Cardinalfish (Pterapogon kauderni): a behavioural experimental approach

The Banggai cardinalfish Pterapogon kauderni is an endangered endemic species with an exceptionally small native distribution and an unusual life history. A paternal mouthbrooder with direct development, symbiosis with benthic organisms (referred to as microhabitat), is crucial to its survival. This is especially so for postflexion larvae (recruits), after their release from the male parent’s buccal cavity. Microhabitat preference in P. kauderni has been studied empirically based on a survey of wild populations. This study adopted an ex-situ experimental approach to P. kauderni microhabitat preference using two well established behavioural trial methods: choice flume and choice tank. The experimental animals were sourced from the introduced P. kauderni population in Palu Bay, Central Sulawesi, Indonesia. The choice flume method was applied at the Central Sulawesi Marine and Fisheries Service Hatchery in Mamboro, Palu, with microhabitats Diadema setosum and D. savignyi. The choice box method was applied at the Universitas Hasanuddin Centre for Research and Development in Makassar, South Sulawesi, Indonesia, with microhabitats Heteractis crispa, Entacmaea quadricolor, D. savignyi and D. setosum. The results indicate the most to least preferred preference hierarchy of D. savignyi, D. setosum, H. crispa, E. quadricolor. While the preference for the sea anemone H. crispa compared to E. quadricolor is consonant with empirical in situ studies on P. kauderni, the observed preference for D. savignyi over D. setosum was unexpected, and points to the need for further research. Observations made during the trials also raise the possibility that imprinting may occur and influence subsequent microhabitat preference.

Previous studies have quantified the relative prevalence of P. kauderni associations with sea urchin and sea anemone species in the wild [14,15,17,18]. The two most common sea urchin P. kauderni microhabitat species are Diadema setosum and D. savignyi [14,17], while in recent studies the most frequently observed sea anemone microhabitats were (in descending order) Actinodendron sp., Heteractis crispa, Entacmaea quadricolor, Stichodactyla gigantea, S. haddoni and H. aurora [18]. Although one experimental study found no apparent preference between D. savignyi and D. setosum in sub-adult and adult P. kauderni [14], there is a lack of experimental studies on microhabitat preference in P. kauderni. One major threat to the endemic P. kauderni populations is the widespread decline in Diadema and sea anemone abundance, due mainly to heavy exploitation, predominantly for human consumption [3,[18][19][20][21].
Behavioural experiments can be used to test hypotheses developed based on empirical (e.g. survey) data; choice flumes and choice tanks are well-established methods in behavioural studies on preference in aquatic animals, including marine fishes [22][23][24][25][26][27][28][29]. The choice tank method (e.g. [27]) and choice flume method (e.g. [28,29]) both present trialled organisms with a choice of attractants within a confined space. Behavioural experiments on P. kauderni microhabitat type preference could provide important information to guide conservation, including habitat and microhabitat rehabilitation. This is particularly relevant within the context of the BCF Gardens concept [30], in which microhabitat protection and, where necessary rehabilitation, are advocated as crucial to the recovery and sustainable management of P. kauderni populations.
This research studied the Banggai cardinalfish -microhabitat symbiosis, in particular the betweenspecies preference of P. kauderni for particular sea anemones and Diadema species, as well as the preference of P. kauderni for sea anemones compared to Diadema urchins. The aim of the experimental behavioural trials was to test for significant preference between microhabitat types in P. kauderni of different age/size classes.

Materials and methods
Experimental animals obtained for the experimental research were Pterapogon kauderni, sea anemones, and Diadema sea urchins ( Table 1). All animals were collected, transported, and handled in compliance with existing regulations and protocols. ---a Introduced P. kauderni population site in Palu Bay, Central Sulawesi [31]; this population appeared to have been extirpated by the 28 September 2018 tsunami in Palu Bay [17] b Barrang Lompo Island (D. setosum) and Badi Island (D. savignyi) In addition to the animals listed in Table 1, specimens of Actinodendron sp. collected in the Spermonde Archipelago were also obtained from ornamental fishermen/traders. However, these highly venomous anemones [32] proved hard to keep in captivity [18] and could not be used in the trials. Separate holding tanks were provided for trialled and untrialled organisms to prevent the same individual from being used more than once during the same trial. These tanks were provided with aeration. Diadema urchins were given seaweed (Ulva sp.) and Enhalus acoroides leaves (collected at the same time as the sea urchins) as feed (ad libitum). P. kauderni were fed on Artemia sp. (cultivated on-site) and/or small mysid shrimp (purchased frozen from an aquarium shop) both before and after the trials and, for the choice tank experiments, during the trials. The anemones were also fed frozen mysid shrimp, delivered close to the oral disc with a pipette. It was noted that the Diadema urchins also consumed mysid shrimp, which settled on the test or accessible areas of the tank floor.
The P. kauderni microhabitat preference trials were carried out from June to August 2018, using two experimental approaches, a choice tank, and a choice flume. Pterapogon kauderni at different lifestages (based on the standard length -SL) were used in the experiments. Based on biological characters, the main life stage classes are recruits (SL ≤ 18 mm, skeleton lacking some elements [33]), juveniles (18 mm < SL < 35 mm), and adults (SL > 40 mm, largest recorded 61 mm) [13]. Fish in the 35-40 mm range may be juveniles, sub-adults with developing gonads, or fully matured adults [13], but in this study were categorized as sub-adults. The microhabitat pairings and number/size classes of P. kauderni trialled using both methods are shown in Table 2. Diadema savignyi vs. none 10 47.6 4.8 A a SD = standard deviation; b R = recruits; J = juveniles; SA = sub-adults; A = adults The choice tank design was adapted from [27], and was constructed and deployed in the Medaka Centre and Water Quality Laboratory, Centre for Research and Development, Hasanuddin University, Makassar, South Sulawesi. The choice tank (Figure 1) was constructed by dividing a polystyrene tank (approximately 45 x 80 cm, internal width around 36 cm) into three sections using movable grids. Microhabitat organisms were placed at either end, with no microhabitat in the central section into which the trialled fish were released.
The placement of the two microhabitat types was exchanged after each replicate to avoid possible end bias, with four replicates per microhabitat pair. Although the microhabitat organisms were confined to their ends by the grids, the grid mesh size was large enough to enable P. kauderni (3 fish per trial replicate) to move freely to all sections of the tank. Observations of P. kauderni microhabitat association were made at intervals over 24 hours (minimum 30 observations per replicate). Water condition and aeration were maintained through intermittent use of a protein skimmer in the centre compartment. In essence, the choice flume method allows large numbers (sufficient to be considered statistically valid, typically around 30) of the organism being tested for preference to be exposed to water flowing past two alternative attractors or repellents to which the tested individual might be expected to respond. While vision may be a factor (depending on flume design), olfaction is usually the main clue [23,34]. Examples of attractors/repellents could be predators [24,35,36], conspecifics [23,26,28], habitat cues [23,28,29,34,37], or microhabitat (e.g. type or condition) [22,24,25,38]. Methods of "infusing" the two water sources with the desired attractant/repellent vary, as do the ways in which water is supplied to and flows out of the flume. In the test chamber, where individuals will be tested for preference, there is no physical barrier to movement; however, the water flow must be laminar (as turbulence would promote mixing) and carry each of the desired signals on just one side of the tank.
The choice flume design was adapted from [23] and A clay pot with a hole about halfway up one side was used for transferring the fish from the holding tanks and releasing them into the choice flume and was fitted with a removable mesh cover. The cover was removed to release the fish after an acclimation period of 1 minute. A stand was constructed of polypropylene piping and fitted with a holder for attaching the GoPro (Hero4) camera. The rate of flow was adjusted to maintain laminar flow without posing a significant challenge or stress to the fish being tested (e.g. well within the range of local tidal currents). Prior to operating the choice flume, the laminar flow was tested using coloured non-toxic (food) dyes, which were fully flushed from the system before trials were commenced.
After acclimation and release, each fish was filmed for 2 minutes (120 seconds). The observer viewed the resulting videos and for each trial noted the time spent by the fish in each of four behaviour categories: POT = fish remaining in or close to the pot; MH1 = fish approaching or remaining close to microhabitat 1 (left of the centreline); MH2 = fish approaching or remaining close to microhabitat 2 (right of the centreline); OT = other (e.g. fish swimming around at the outflow end of the flume). MH1 and MH2 could be D. setosum, D. savignyi, or none, depending on the trial.

A B
The  B. Screenshot taken from the GoPro camera used to film each trial when fixed in place with microhabitat organisms (in this case, Diadema setosum left and D. savignyi right) confined to compartments at the upstream end. Trialled fish were placed in the pot (initially covered for 1-minute acclimation) and filmed for 2 minutes (120 seconds) once the cover was removed.
The choice tank and choice flume data were tabulated in Microsoft Excel 2010. For each set of trials, Pearson's chi-square test (chisq.test) in R version 3.4.2 [39], implemented in RStudio version 1.1.456 [40], was applied to test the null hypothesis of no preference between microhabitat types.

Choice tank trials
The preferences recorded in the choice tank experiments (Table 3) were mostly conducted with subadult/adult P. kauderni. The results indicate a significant preference for microhabitats compared to no microhabitat, and most fish moved several times between microhabitats. Diadema savignyi was significantly preferred to Diadema setosum, in contrast to [14], who found no significant preference. With regards to H. crispa versus either of the two Diadema species, despite an overall preference for Diadema, H. crispa was preferred to D. savignyi in a trial replicate with juvenile fish (SL < 35 mm). Of the four trials, preference between the two anemone species had the lowest p-value (p < 0.05); however, some preference for H. crispa to E. quadricolor is consonant with field survey results [18].

Choice flume trials
The results of the choice flume experiments carried out at Mamboro (Table 4, Figure 3) indicate a significant preference in P. kauderni of all size classes for sea urchins over no microhabitat and for D. savignyi over D. setosum. However, it was remarkable that the seven P. kauderni recruits collected

Implications for conservation management
In contrast with the results in Tables 3 and 4, an earlier behavioural experiment using different methods [14] found no significant difference in sub-adult/adult P. kauderni association with (equal numbers) of D. savignyi and D. setosum. These contradictory results call for further investigation. With respect to the P. kauderni size class, the fish in [14] were sub-adult to adult P. kauderni (35)(36)(37)(38)(39)(40)(41)(42). Collectively, the choice flume and choice box trials cover a full range of P. kauderni size classes, with recruits, small juveniles, and adults in the choice flume trials, and larger juvenile and sub-adult P. kauderni in the choice tank trial of D. setosum vs. D. savignyi. One possible explanation is that in the large concrete tanks used by [14], any preference was confounded by the tendency of the sea urchins to congregate in mixed-species groups, while in this study, the two Diadema species were kept separate. Conversely, despite the high statistical significance of the observed preference for D. savignyi in this study, it is still possible that our results could be due to chance.
Diadema savignyi and D. setosum appear to have overlapping, but significant distributions across habitat types, and thus together may enlarge the potential habitat for P. kauderni [17]. While increased exploitation of Diadema spp., mostly for human consumption, is the main driver of declining urchin populations across much of the P. kauderni endemic range [3,[18][19][20][21], it is not known whether collection affects both species proportionally or not. Captive breeding has not been developed for either Diadema species. In addition, analysis of mitochondrial DNA (mtDNA) has shown that Diadema clarki mtDNA is also present in the Banggai Archipelago, possibly as a full species, in hybrids or through introgression [17]. It is clear that there is still much to learn about the P. kauderni -Diadema symbiosis; however, the study results support the view that D. savignyi is important as a P. kauderni microhabitat and that efforts to rehabilitate Diadema microhabitat should aim to maintain both species rather than concentrating on the generally more abundant D. setosum.
To some extent, the frequency of P. kauderni association with different sea anemone species recorded during a field survey in 2017 [18] may have been due to availability as much as preference. For example, the host sea anemone Stichodactyla gigantea (fourth most common host) was rare or absent from most sites [18]. Visual records Ndobe unpublished data, 2004-2006) indicate that this anemone was once more prevalent, both in terms of absolute abundance and as a P. kauderni microhabitat. In the Banggai Archipelago, this large and fleshy anemone has been more heavily collected (mostly for human consumption) than any other anemone, especially since 2007 [3,7]. This species is also collected for the marine ornamental trade. For example, in the Spermonde Archipelago, Indonesia [44] and the Philippines [45], and collection may well extend to the Banggai Archipelago.
By comparison, both H. crispa and E. quadricolor are still relatively abundant in the Banggai Archipelago. Heteractis crispa is both easier to propagate asexually than E. quadricolor [18] and appears to be preferred as a microhabitat in the wild [18] and under experimental conditions (Table 3). These findings indicate that H. crispa could be a primary target species for microhabitat rehabilitation to promote P. kauderni population recovery, as proposed in the BCF Garden concept [2,30].
The results indicate that there may be factors other than ontogeny influencing microhabitat preference. For example, as is thought to occur in clownfishes of the genus Amphiprion [38,[41][42][43], imprinting may play a significant role in P. kauderni microhabitat preference. In addition, it has been postulated that imprinting may affect the choice of egg-laying sites in clownfishes [42]. The ambiguous (and in some cases conflicting) data on microhabitat preference point to a possible role of imprinting is in P. kauderni microhabitat choice. It is not impossible that imprinting of recruits could affect microhabitat choice at later life stages, in particular the choice of recruit release sites selected by