Pipefish

Two new and the only known extinct seahorse species Hippocampus sarmaticus and Hippocampus slovenicus are described from the Middle Miocene beds (Lower Sarmatian) in Slovenia, representing the oldest known fossil record of seahorses. Hippocampus sarmaticus was most similar to the extant seahorse species Hippocampus trimaculatus, while H. slovenicus can be most easily compared to the extant pygmy seahorses H. bargibanti, H. denise, and H. colemani. These Sarmatian seahorses lived among seagrasses and macroalgae in the temperate shallow costal waters of the western part of the Central Paratethys Sea.

Background. Syngnathid fishes produce three kinds of sounds, named click, growl and purr. These sounds are generated by different mechanisms to give a consistent signal pattern or signature which is believed to play a role in intraspecific and
interspecific communication. Commonly known sounds are produced when the fish feeds (click, purr) or is under duress (growl).While there are more acoustic studies on seahorses, pipefishes have not received much attention. Here we document the differences in feeding click signals between three species of pipefishes and relate them to cranial morphology and kinesis, or the sound-producing mechanism.

Methods. The feeding clicks of two species of freshwater pipefishes, Doryichthys martensii and Doryichthys deokhathoides and one species of estuarine pipefish,
Syngnathoides biaculeatus, were recorded by a hydrophone in acoustic dampened tanks. The acoustic signals were analysed using time-scale distribution (or scalogram) based on wavelet transform. A detailed time-varying analysis of the spectral contents of the localized acoustic signal was obtained by jointly interpreting the oscillogram, scalogram and power spectrum. The heads of both Doryichthys species were prepared for microtomographical scans which were analysed using a 3D
imaging software. Additionally, the cranial bones of all three species were examined using a clearing and double-staining method for histological studies.

Results. The sound characteristics of the feeding click of the pipefish is speciesspecific, appearing to be dependent on three bones: the supraoccipital, 1st postcranial plate and 2nd postcranial plate. The sounds are generated when the head of the Dorichthyes pipefishes flexes backward during the feeding strike, as the supraoccipital slides backwards, striking and pushing the 1st postcranial plate against (and striking)
the 2nd postcranial plate. In the Syngnathoides pipefish, in the absence of the 1st postcranial plate, the supraoccipital rubs against the 2nd postcranial plate twice as it is pulled backward and released on the return. Cranial morphology and kinesis
produce acoustic signals consistent with the bone strikes that produce sharp energy spikes (discrete or merged), or stridulations between bones that produce repeated or
multimodal sinusoidal waveforms.

Discussion. The variable structure of the sound-producing mechanism explains the unique acoustic signatures of the three species of pipefish. The differences in cranial
bone morphology, cranial kinesis and acoustic signatures among pipefishes (and seahorses) could be attributed to independent evolution within the Syngnathidae,
which warrants further investigation.

Calcein marking via osmotic induction and overnight immersion was compared in saltwater fishes. Immersion in a salt solution for 10 min followed by 30 min in a 500 mg l−1 calcein solution produced the highest fluorescent mark in 40 min with no effect on survival.

An account of the first documented spawning of the pipefish Microphis aculeatus (Syngnathidae) is presented. Pipefishes and seahorses are sex-reversed and males brood ova, displaying a level of parental investment extraordinary among teleost fishes. Pipefishes are known to exhibit complex courtship rituals, described here for M. aculeatus from a series of spawning events in a captive population at a public aquarium. Reproductive individuals ranged from 125-145 mm (male) and 136-144 mm (female) in total length giving a first estimate of size at maturity for the species. Spawning events (n=19) occurred in November over a period of 10 days immediately following illumination of the exhibit, suggesting that lighting is a primary cue in reproductive behaviour for this species. The courtship behaviour is initiated by males and can be divided into five distinct phases (described herein) which are characterised by displays of body quivering, rostral pointing, caudal fin fanning and curvature of the body into a distinctive "S" shaped curve while displaying the ventral side (harbouring the brood pouch) towards the female. Respiration rates were markedly increased during copulatory behaviour, peaking at 128 min-1 (male) and 120 min-1 (female). The observations of breeding and ventilation rates have implications for the nutritional status, age class and general husbandry practices required for spawning these pipefishes in an aquarium.