Forensic biological pursuits of exotic fish origins: piranha in Hawaii

Synopsis The otoliths of an adult red-bellied piranha, Pygocentrus nattereri, captured from a reservoir in Hawaii were scrutinized to determine the fish's origin and growth history. Sagittal otoliths of the piranha, P. nattereri, contained internal microincrements visible by Scanning Electron Microscopy. The medial cross sectional plane of the sagitta was resolved to provide counts for the most visible micro-increments. An opportune spawning of confiscated adults provided samples for verification of daily increment formation. Daily formation of microincrements was verified from hatched individuals, and confirmed the suitability of otoliths for revealing daily patterns in the age and growth of piranhas. The central area of the sagitta was diffuse in regards to otolith microstructure and indicated the fish was held in an unchanging environment (aquarium). Therefore, otoliths provide important life history or forensic information incorporated within their structural components. The visualization of daily microincrements in the otolith of a juvenile allowed the determination of age at and since release. Fish grew rapidly after being released into the wild. From otolith increments the date of release for an individual fish can be calculated with acceptable accuracy. As presented, otolith structural information can provide age and growth data which are essential to the management of introduced species.     

Growth and morphological development of sagittal otoliths of larval and early juvenile Trachurus japonicus

The daily periodicity of growth increment formation in sagittal otoliths of jack mackerel Trachurus japonicus was validated by marking otoliths with alizarin complexone (ALC). Analysis of otoliths of known‐age juveniles confirmed that the first increment formed on day 3 after hatching, and was associated with first feeding. A total of 198 specimens, ranging from 2·6 to 49·2 mm in body length (notochord length or standard length) and from 7 to 78 days in age, were collected in the East China Sea and Tosa Bay, and used to examine the association between otolith morphological development and ontogenetic development. The relationship between body length ( L ) and otolith radius ( R ) was significantly described by the linear function L  = 2·65 + 0·0425 R ( n  = 198, r ² = 0·99, P  < 0·001), indicating that somatic growth history can be reconstructed from otolith growth patterns. The otolith was primarily spherical in the preflexion larval stage, and became elongated with notochord flexion. The first secondary primordium formed at c . 25 days, during the middle postflexion stage, and was associated with metamorphosis. By c . 42 days the sagittal otolith was adult‐like in morphology, with the primary growth zone enclosed by the marginal growth zone, except in the anterior rostrum area. Thus age, growth and developmental stages were recorded in sagittal otoliths during the larval and early juvenile stages of jack mackerel.     

Validation of daily microincrement deposition in otoliths of juvenile and adult Peruvian anchovy Engraulis ringens

Wild adult specimens of the Peruvian anchovy Engraulis ringens were captured and reared to validate the daily periodicity of otolith microincrement formation. The postcapture stress generated spontaneous spawning, making it possible to conduct a rearing trial on larvae first in an artificial nutrient‐enriched system (ANES) for 52 days followed by an artificial feeding regime in a culture tank until day 115 post‐hatch. Microincrements of the sagittal otoliths of sacrificed juveniles [mean ± s.d. total length (L_T) = 5·13 ± 0·37 cm, range 5–6 cm; c.v. = 7·5%] showed very distinct light and dark zones. The slope of the relationship between the total number of increments after the hatch check and days elapsed after hatching was not significantly different from 1. The transfer from ANES to the artificial feeding regime induced a mark in the sagittal otoliths. The number of microincrements after this induced mark coincided with the number of days elapsed after the transfer date. In parallel experiments, adult E. ringens (mean ± s.d. L_T = 14·92 ± 0·55 cm, range 13–16 cm) were exposed to one of two fluorescent marking immersion treatments with either alizarin red S (ARS; 25 mg l^?1 per 6 h) or oxytetracycline hydrochloride (OTC; 200 mg l^?1 per 10 h). The microincrements between fluorescent bands were distinct, ranging from 0·89 to 2·75 µm (mean ± s.d. =1·43 ± 0·28 µm; c.v. = 32%) and from 0·71 to 2·89 µm (1·53 ± 0·27 µm; c.v. = 35%) for ARS and OTC, respectively. The relationship between the number of microincrements between marks and the number of elapsed days for ARS and OCT treatments indicated that there was a significant correspondence between the number of increases observed and the number of days. Hence, daily microincrements of otoliths of E. ringens are likely to be formed in juveniles and adults under natural conditions.     

Validation of daily otolith increments in larval and juvenile Chinese sucker, <Emphasis Type="Italic">Myxocyprinus asiaticus</Emphasis>

Otolith development was observed and the formation of daily growth increments in otoliths of Chinese sucker, Myxocyprinus asiaticus, was validated by monitoring known-age larvae and juveniles in the laboratory from 2003 to 2005. Otolith shape changed with larval and juvenile development, and there was an exponential relationship until a body length of 16 mm or so, and a linear relationship after a body length of 16 mm between otolith size and fish size. The first increment was identified in larvae 1 day after hatching. The regressed equations between daily age (D) and increment number in otoliths (N) were N = −0.64 + 0.96D in lapillus, and N = −0.31 + 0.98D in sagitta. The slopes were not significantly different than 1.0. This demonstrated that otolith increments in this species were formed daily and can be used for daily age determination.     

Otolith microstructure of <Emphasis Type="Italic">Oxygymnocypris stewartii</Emphasis> (Cypriniformes,Cyprinidae, Schizothoracinae) in the Lhasa River in Tibet,China

Otolith microstructure of Oxygymnocypris stewartii collected from the Lhasa River was examined and described with regards to the early life history events. The monthly changes in the number of microincrements on the margin of the otolith were examined to validate the approximately daily periodicity of otolith increment formation. The microstructure of otoliths was used to detect changes in microincrement deposition patterns corresponding with events during early life. The annuli, microincrements and checks including the hatch check, yolk absorption check and several recurrent patterns in the otolith were described. Periodicity of the recurrent patterns was weekly, fortnightly and monthly. Through counting the number of the microincrements, it was confirmed that the primary growth of O. stewartii was in a period of 7 or 8 months from late March to October; it was estimated that O. stewartii might hatch between April and May.     

Otolith shape analysis and daily increment validation during ontogeny of larval and juvenile European chub Squalius cephalus

This assesses features of otoliths from laboratory-reared embryos, larvae and juvenile European chub Squalius cephalus from hatching to 180 days post-hatching (dph). We observed the development of the three pairs of otoliths (lapilli, sagittae and asterisci) and more precisely shape changes, as well as timing and deposition rate of increments of the lapilli. The lapilli and the sagittae were present at hatching, whereas the asterisci formed between 20 and 30 dph. The lapillus and sagitta shapes were round until 20 dph. From 60 dph the anterior and the posterior rostra of the sagittae were well developed, but very thin, making this otolith too fragile to manipulate for further studies of shape and validation of otolith increment deposition rate. The lapilli provided reliable age estimates for free embryos, larvae and juveniles up to 120 dph. However, caution should be taken when ageing fish older than 150 dph as an underestimation was noticeable. The regression of the number of otolith increments on age showed a slope and an intercept not significantly different from 1 and 0, respectively, which indicated that otolith growth increments were deposited on a daily basis, with the first microincrement occurring at hatching. Increment counts were consistent between three interpreters, indicating a consistent and reliable age estimate. This study validates that the otolith increment deposition rate can be used to assess hatching dates and daily growth of wild S. cephalus under 150 dph and in environments similar to the conditions used in this study.     

Age and early life history of juvenile scotia sea icefish, <Emphasis Type="Italic">Chaenocephalus aceratus</Emphasis>, from Elephant and the South Shetland Islands

The otolith microstructure of juvenile Scotia Sea icefish (Chaenocephalus aceratus) was analyzed from samples collected around Elephant and South Shetland Islands, with the aim to validate previous annual ageing and to give new insight into its early life history timings. Fish were caught by bottom trawl fishing conducted on the continental shelf between 100 and 500 m depth. To determine the timing and position of the first annulus on sagittal otoliths, microincrements were counted on juvenile otoliths previously aged 1+ year old by counting annuli in sectioned otolith. Assuming that microincrements were laid down daily, age ranged from 406 to 578 days in fish measuring 13–19 cm TL, thus corroborating previous results. The relationship between fish size and otolith size/weight was estimated using the least square linear regression method. The relationship between age and otolith size was also estimated to determine the otolith length in 1-year old fish, which was approximately 1.58 mm. In all samples the otolith core was characterized by an evident strong check, assumed to be laid down at the beginning of exogenous feeding of yolk sac larvae. The yolk sac duration estimated from hatch to the first feeding check was longer than other channichthyids, lasting 29–45 days. Hatching dates were backcalculated from the date of capture using the age estimates, indicating C. aceratus sampled off Elephant and South Shetland Islands hatched over a long period lasting from July to December, with a peak in November. As a result, the potential larval dispersion driven by local oceanographic features is discussed.     

Historical changes in juvenile southern bluefin tuna Thunnus maccoyii growth rates based on otolith measurements

Suspected historic changes in juvenile southern bluefin tuna Thunnus maccoyii growth rates were investigated using otolith increment width data. Four hundred and ninety otoliths were selected from fish estimated to be between 1 and 41 years-old. The distance between the first five annuli were measured on the otoliths, giving estimates of otolith growth for age classes 1+ to 4+ years for fish spawned from the early 1960s to mid 1990s. The data showed that growth rates of juveniles (age 1+ and 2+ years) started to increase at around 1979–1980, and that growth continued to increase throughout the 1980s and early 1990s. Lee's phenomenon was not observed in the data. Correlation tests did not reveal clear relationships between annual otolith growth and regional environmental variables such as sea surface temperature or Southern Oscillation Index. The increase in otolith growth, however, was consistent with juvenile growth estimates obtained from other sources, and correlated with large-scale trends in population size and environmental conditions.     

Validation of daily increment formation in otoliths for three Diplodus species in the Mediterranean sea

The efficacy of two fluorochromes, chlorhydrate of tetracycline (CHTC) and alizarin complexone (ALC), to induce a label on the otoliths of juvenile sparid fishes by immersion techniques was tested in different experimental conditions. CHTC did not mark otoliths in natural sea water and was toxic in divalent cation-free sea water. Immersing fish in a 50-mg 1⁻¹ ALC natural seawater solution for 24 h induced a well-defined mark on the otoliths and had no effect on survival. A daily periodicity of increment formation on otoliths was observed in captivity for Diplodus vulgaris and D. puntazzo , and was conserved in natural environment for D. sargus . The frequency of increment deposition did not vary with the age of juvenile fishes. Thus, otolith microstructure analysis will be a reliable method to give age estimates in these three sparid species during their juvenile life.     

Environmental effects on primary increment formation in the otoliths of newlyhatched Arctic charr

Otolith microincrements were investigated in Arctic charr Salvelinus alpinus , reared from hatching under various temperatures (1, 3, 5, 7° C) and feeding conditions (starved, fed every third day, fed daily). Larval charr otoliths were marked with oxytetracycline and alizarin complexone. Alizarin complexone was found to be 100 per cent successful in marking otoliths while oxytetracycline marks could be seen in <10 per cent of the otoliths viewed. Otolith microincrements were viewed by light and scanning electron microscopy to investigate the daily nature of increment deposition. Low temperatures (1 and 3° C) and starvation depressed daily increment formation. Increment deposition was found to be daily among the larvae reared at warmer temperatures (5 and 7° C) and fed at least every third day. Scanning electron microscopic analysis allowed us to confirm the results of light microscope increment counts from all temperatures except 1 ° C, where the number of increments enumerated were higher than the number obtained during light microscopy analyses. Increased feeding and warmer temperatures also resulted in increased increment width. The difference in increment number and width seems to be dependent upon fish growth rate which we have found to be affected by both temperature and feeding conditions.     

Otolith formation,microstructure and daily increment validation in juvenile perch Perca fluviatilis

The otoliths of laboratory‐reared larval and juvenile perch Perca fluviatilis of known age were analysed to determine the age of otolith formation and validate the formation of daily increments. There was a linear relationship between number of increments and age in days post‐hatching, although by 82 days post‐hatching daily increment counts underestimated actual age by an average of 5 days. Otolith dimensions in relation to standard length indicated allometric growth of otoliths until completion of yolk absorption, and isometric growth thereafter, up to 82 days post‐hatching.     

Morphometric analysis of otoliths of juvenile crucifix sea catfish Sciades proops (Valenciennes, 1840)

The objective of this study was to analyze the morphometry of otoliths for Sciades proops juveniles by testing the hypothesis of equality in morphometric relationships for the right and left otoliths, which could then be interchangeably used to estimate fish size or weight. Samples were obtained monthly directly from anglers after each event that took place off the state of Sergipe from March/2014 to April/2015. Anglers used rod and reel during these events, with no restriction on hook size or line thickness. Each fish specimen sampled had their total weight (W, g) and total length (TL, cm) measured and their lapillus otoliths removed and stored separately. Each otolith had its length (OL), width (OWi), and thickness (OT) measured (all in mm) under a stereomicroscope. Otoliths were weighed using a precision scale (OW, g). A total of 883 specimens were sampled: TL = 12.0–60.5 cm and W = 9.8–1880 g. The weight‐length relationship for the juvenile fishes was W = 0.0052TL^3.086 and for their otoliths was OW = 0.0002OL^3.177. The weight‐length and length‐length relationships fitted for each otolith (right and left) were not statistically different and thus all relations were estimated for grouped otoliths. The length‐length relationships for the otoliths were: OWi = 0.947OL?0.205 and OT = O.484OL?0.698. The relationship estimated for juvenile fish and otolith weight was Wj = 1076.1OW?9.120. For juvenile fish total length and otolith length, width and thickness, the following relationships were estimated: TLj = 4.028OL?3.199, TLj = 4.208OWi?2.091, and TLj = 7.824OT + 3.659, respectively. Relationships between fish and otolith size, and between fish and otolith weight indicated a change in slope close to L_m50, which should be better explored when more adult specimens are available.     

All in the ears: unlocking the early life history biology and spatial ecology of fishes

Obtaining biological and spatial information of the early life history (ELH) phases of fishes has been problematic, such that larval and juvenile phases are often referred to as the ‘black box’ of fish population biology and ecology. However, a potent source of life‐history data has been mined from the earstones (otoliths) of bony fishes. We systematically reviewed 476 empirical papers published between 2005 and 2012 (inclusive) that used otoliths to examine fish ELH phases, which has been an area of increasing attention over this period. We found that otolith‐based research during this period could be split into two broad themes according to whether studies examined: (i) biological objectives related to intrinsic processes such as larval and juvenile age, growth and mortality, and/or (ii) spatial objectives, such as habitat use, dispersal and migration. Surprisingly, just 24 studies (5%) explored a combined biological–spatial objective by simultaneously exploiting biological and spatial information from otoliths, suggesting much more scope for such integrated research objectives to be answered via the use of multiple otolith‐based techniques in a single study. Mapping otolith analytical techniques across these two approaches revealed that otolith structural analysis was mainly used to investigate biological processes, while otolith chemical analyses were most often applied to spatial questions. Heavy skew in research effort was apparent across biomes, with most (62%) publications specific to marine species, despite comparable levels of species richness and the importance of freshwater taxa (just 15% of papers). Indeed, around 1% (380 species) of a possible 31400+ extant species were examined in our surveyed papers, with a strong emphasis on temperate marine species of commercial value. Potential model species for otolith‐based ELH ecology research are arising, with the eel genus Anguilla (24 studies) and the European anchovy Engraulis encrasicolis (14 studies) attracting more research effort than most other taxa. While there is a preponderance of common techniques (e.g. daily otolith increment counts, increment widths), novel techniques such as transgenerational marking and computed X‐ray tomography, are increasingly being applied in published studies. The application of an integrative approach based on a combination of emerging techniques and traditional methods holds promise for major advances in our understanding of ELH fish ecology and to shine light into the ‘black box’ of fish ecology.     

Factors affecting morphological development of the sagittal otolith in juvenile and adult small yellow croaker (Larimichthys polyactis Bleeker, 1877)

The morphology and morphometrics of the sagittal otoliths of small yellow croaker (Larimichthys polyactis) from the southern Yellow Sea were investigated. Study objectives were to evaluate the shape variability and morphometric variables of sagittae of juveniles and adults as related to developmental changes and habit shift. A total of 152 fish were sampled from April to June of 2012 and 2013, along the coastal waters of the Lüsi spawning ground in the southern Yellow Sea. Changes in otolith shapes from the juvenile to the adult were presented with the rim development through the entire‐lobed‐entire transition and with the curvature of the cauda toward the ventral margin. The otolith elongation in the juvenile stage occurred at 10–20 mm standard length (SL) and was likely associated with the formation of otolith accessory growth centers from larvae to juvenile. The L. polyactis sagittal otoliths acquired their definitive shape at 130 mm SL maturity. Ontogeny on otolith shape might be related, for example, to the factors of metamorphic development, feeding habitat, and ambient water salinity, which varied throughout the growth of L. polyactis.     

Early otolith development in the critically endangered tooth-carp, <Emphasis Type="Italic">Aphanius farsicus</Emphasis> (Teleostei: Cyprinodontidae)

Otolith morphology in the tooth-carp/killifish genus Aphanius is a source of informative taxonomic characters at both the species and population level. Most work on otoliths has focused on adult specimens, while evidence of ontogenetic variation is rarely provided. In this study we describe the development of otolith morphology during the early life stages of an endangered and endemic species, the Fars tooth-carp Aphanius farsicus from southern Iran. The study material comprises 34 larvae and early juveniles representing nine different developmental stages (0–120 days post hatching), all reared under the same laboratory conditions. The results reveal (i) a significant correlation between standard length and otolith size (length) in larval and early juvenile stages, (ii) clear differences in otolith morphology between larvae/early juveniles and adults, and (iii) a temporal link between the appearance of the sulcus on the otolith’s inner face and the emergence of the dorsal and anal fins. Our results indicate that otoliths of Aphanius can be recognized as originating from larval or early juvenile fish based on their short rostrum and antirostrum lengths and wide excisura, in addition to their small size. These immature otoliths are, however, not diagnostic at the species level in A. farsicus, nor most probably in other species of tooth-carp. The outcome of our study is also of interest to palaeontologists working with fossil killifish otoliths, as it can help avoid misinterpretation of ancient species diversity.     

三峡库区木洞江段翘嘴鲌早期生长特征研究

2013 年 910 月在三峡库区木洞江段采集翘嘴鲌(Culter alburnus)幼鱼, 摘取微耳石进行耳石微结构分析, 推算了翘嘴鲌幼鱼的日龄及孵化日期, 探讨其早期生活史阶段的生长特征。结果显示, 采集 97 尾翘嘴鲌幼鱼, 体长范围为 4098 mm。翘嘴鲌幼鱼的微耳石形状为不规则扁椭球形, 耳石横截面磨片上具有一个核和一个原基。耳石原基的直径为11.627.8 m, 平均值为(18.63.8) m。耳石核中心到第一个生长轮的距离为(13.04.7) m。翘嘴鲌幼鱼的日龄为 44104d, 推算其孵化日期为 2013 年 6 月 9 日至 8 月 17 日, 高峰期为2013 年7 月9 日至7 月22 日。耳石半径与体长、日龄与体长之间均呈显著的线性关系(P0.05)。耳石日轮宽度随着日龄的增加不断变化显示, 三峡库区翘嘴鲌早期生活史阶段的生长速率不断变化, 日平均生长率为0.774 mm/d。       

Morphometry and composition of aragonite and vaterite otoliths of deformed laboratory reared juvenile herring from two populations

Vaterite otoliths were sampled from two reared populations (Celtic and Clyde Seas) of juvenile herring Clupea harengus. The crystallography, elemental composition and morphometry were analysed and compared with those of normal aragonite otoliths. The incidence of vaterite otoliths in the juveniles sampled (n = 601) ranged from 7·8% in the Clyde population to 13·9% in the Celtic Sea population, and was 5·5% in the small sample (n = 36) of wild adults examined. In all but one case fish had only one vaterite otolith; the corresponding otolith of the pair was completely aragonite. Although the majority of the juveniles sampled showed craniofacial deformities, there was no link between the skull or jaw malformation and the incidence of vaterite otoliths. All vaterite otoliths had an aragonite inner area, and vaterite deposition began sometime after the age of 90 days. The vaterite otoliths were larger and lighter than their corresponding aragonite partners, and were less dense as a consequence of the vaterite crystal structure. The vaterite areas of the otoliths were depleted in Sr, Na and K. Concentrations of Mn were higher in the vaterite areas. The transition between the aragonite inner areas and the vaterite areas was sharply delineated. Within a small spatial scale (20 μm³) in the vaterite areas, however, there was co‐precipitation of both vaterite and aragonite. The composition of the aragonite cores in the vaterite otoliths was the same as in the cores of the normal aragonite otoliths indicating that the composition of the aragonite cores did not seed the shift to vaterite. Vaterite is less dense than aragonite, yet the concentrations of Ca analysed with wavelength‐dispersive spectrometry (WDS) were the same between the two polymorphs, indicating that Ca concentrations measured with WDS are not a good indicator of hypermineralized zones with high mineral density. The asymmetry in density and size of the otoliths may cause disruptions of hearing and pressure sensitivity for individual fish with one vaterite otolith, however, the presence of vaterite otoliths did not seem to affect the growth of these laboratory reared juvenile herring.     

An accurate tool for ageing Two‐spined Blackfish for use in determining timing of spawning in upland streams in the Brindabella ranges,ACT, Australia

To manage populations of threatened fish species in modified habitats and regulated rivers requires an understanding of their reproductive biology and spawning cues. In particular, accurate information about early life stages in these species can be used to facilitate programmes that maximise recruitment and breeding success. This study aimed to develop methods to accurately age early juvenile freshwater native fish, Two‐spined Blackfish, (Gadopsis bispinosus), to allow the determination of spawning date. This was accomplished through the examination of otolith microstructure in early juveniles. The age at which the first ring was deposited and the relationship between days and number of rings were determined using both field and aquarium trials. Field trials of marked juvenile otoliths revealed daily deposition of rings (1.02 ± 0.02 rings per day for fish sacrificed six days postmarking). The strength of this relationship lessened slightly as juveniles aged (0.92 ± 0.02 rings per day for fish sacrificed 13 days postmarking). The first otolith ring was deposited 7.50 ± 1.09 days after spawning. The enumeration of daily rings combined with knowledge of the commencement of ring deposition enabled accurate estimation of spawning date. The current study is the first to examine otolith microstructure in juvenile Two‐spined Blackfish allowing accurate determination of spawning date. While more research is required to accurately age older juveniles, this technique has the potential to precisely correlate spawning with environmental cues, facilitating better management of temperature and flow during breeding periods, potentially increasing spawning and recruitment of this endangered species.     

The detection of elements in larval otoliths from Atlantic herring using laser ablation ICP-MS

Trace element concentrations of otoliths from larval herring Clupea harengus collected from known spawning beds in the Celtic and Irish Seas, were investigated using laser ablation ICP-MS and compared with concentrations in the larval cores of juvenile otoliths from the same populations and year class. A range of elements (Mg, Zn, Sr, Ba and Pb) was detectable in early larval otoliths (20–40 µm diameter). Larval otolith concentrations exceeded the larval core concentrations of juvenile otoliths and also the concentrations reported in the literature, for Mg, Zn, Ba and Pb, indicating that the measurement of elements in larval otoliths was severely affected by post-mortem contamination, most likely due to adherence of tissue and endolymph residue on the otolith surface. Comparison of otolith composition between larvae from two freezing treatments showed that contamination from Mg and Zn was more serious in otoliths that had remained in frozen larvae for prolonged periods. Larval populations from the two seas showed significant differences in otolith Sr concentrations, which were consistent over two sampling years. Similar differences were seen in the corresponding juvenile populations. The results show that while early larval otoliths are extremely susceptible post-mortem contamination, Sr concentrations can be reliably measured using laser ablation ICP-MS and for this element, the detection of region specific differences is possible.     

An evaluation of the otolith characteristics of Conger conger during metamorphosis

A sample of 20 metamorphosing conger eel Conger conger leptocephali were collected from the Minho River, Portugal, in February 1999 and their sagittal otoliths were analysed by scanning electron microscopy. Four different etching agents were applied along both sagittal and frontal sections during otolith preparation to examine the microstructural growth in this species. Otolith growth increments were visible throughout the increment countable zone using all four treatments, but a permanent peripheral diffuse zone, where the daily increments were unclear, appeared on all otoliths, preventing accurate age estimation. To understand more about the nature of the diffuse zone, otoliths of 10 other metamorphosing leptocephali reared in aquaria were marked by immersion in tetracycline hydrochloride. The distance between the fluorescent marks and otolith edge, measured over a fixed period of time, was used to estimate the otolith growth rate. The application of this technique led to an anomalously high estimated otolith growth rate, probably as a result of the capture, marking and handling stress.