Body shape variation in two species of darters (Etheostoma,Percidae) and its relation to the environment

Environmental variation can shape phenotypic variation in organisms, but most evidence for trait differentiation comes from analyses of dichotomous habitat types that differ in only one or few key environmental factors. In reality, environmental variation is often more subtle, gradual and multifarious. Here, we investigated geographic variation in body shape of two darter species (Etheostoma spectabile and Etheostoma flabellare; Percidae) that occur along river gradients. This study addressed three specific questions: Is there intraspecific geographic variation in the two species across different sites in the Ozark Highlands of Oklahoma (USA)? Is phenotypic variation across sites correlated with abiotic environmental conditions? Do the two species share site‐specific (i.e. convergent) phenotypic variation in areas where they occur together? Our results indicated significant body shape variation in both species. Population differences in body shape were particularly correlated with variation in substrate composition. The combined analysis of both species indicated a small but significant effect of convergence on body shape wherever they are sympatric; shared variation, however, was not related to any environmental variables included in the analysis. While it remains unclear whether phenotypic variation in these species is due to heritable differentiation or environmentally induced plasticity, our results indicate that even subtle and gradual environmental variation can induce substantial variation in phenotypes on a relatively small spatial scale.     

Comparative functional responses of native and high‐impacting invasive fishes: impact predictions for native prey populations

Comparative functional responses (FRs) can predict impacts of invasive species, including piscivorous fishes, via quantifying their depletion of native food resources as a function of prey density. The utility of FRs for predicting impacts on prey populations by invasive fishes of different trophic guilds was tested here by comparing the FRs of the invaders Cyprinus carpio and Carassius auratus, with three native, trophically analogous fishes, Barbus barbus, Squalius cephalus and Tinca tinca. Chironomid larvae and Gammarus pulex were used as prey items. Predictions, developed from studies on the foraging of C. carpio and C. auratus in the literature, were that the invaders would have significantly higher consumption rates for chironomids than the native fishes, but not for G. pulex. Mean consumption rates for chironomids were significantly lower for both invaders than B. barbus and S. cephalus, but were similar to T. tinca. Barbus barbus had a significantly lower consumption rate of G. pulex than both invaders, but there were no significant differences between S. cephalus, T. tinca and the invaders. All FRs were type II, with FR curves for the invaders preying upon chironomids never being significantly higher than the native fishes, contrary to predictions. For G. pulex, some significant differences were apparent between the invaders and native fishes, but again were contrary to predictions. These results indicated that when predation impacts of invasive fishes could also be a function of their population density and body sizes, these parameters should be incorporated into FR models to improve impact predictions.     

Protective efficacy of Nigella sativa seeds and oil against columnaris disease in fishes

Columnaris disease, caused by the bacterium Flavobacterium columnare, is currently the most frequently reported bacterial disease affecting farm‐raised channel catfish in the USA. Common treatments against the disease include the use of medicated feed that has led to emergent antibiotic resistant strains of F. columnare. Nigella sativa (Black cumin) is a medicinal herb commonly used by many cultures as a natural remedy for numerous disorders. Recently, we have discovered the antibacterial activity of N. sativa and its oil extract against F. columnare. In this study, we showed N. sativa oil (NSO) strongly inhibited the growth of all of the strains of F. columnare tested and yielded significantly larger zones of inhibition than those produced by oxytetracyclin. We tested the protective effect against columnaris disease in vivo by incorporating NSO (5%) or N. sativa seeds (NSS) (5%) into fish feeds. Fishes (Ictalurus punctatus and Danio rerio) fed amended diets displayed significantly lower mortality than those fed control diets. Per cent mortalities in control groups ranged from 77% to 44% and from 70% to 18% in zebrafish and channel catfish, respectively. A dose study using different NSS concentrations showed that 5% NSS offered the most protection against columnaris disease in channel catfish.     

Cleaner fishes and shrimp diversity and a re‐evaluation of cleaning symbioses

Cleaning symbiosis has been documented extensively in the marine environment over the past 50 years. We estimate global cleaner diversity comprises 208 fish species from 106 genera representing 36 families and 51 shrimp species from 11 genera representing six families. Cleaning symbiosis as originally defined is amended to highlight communication between client and cleaner as the catalyst for cooperation and to separate cleaning symbiosis from incidental cleaning, which is a separate mutualism preceded by no communication. Moreover, we propose the term ‘dedicated’ to replace ‘obligate’ to describe a committed cleaning lifestyle. Marine cleaner fishes have dominated the cleaning symbiosis literature, with comparatively little focus given to shrimp. The engagement of shrimp in cleaning activities has been considered contentious because there is little empirical evidence. Plasticity exists in the use of ‘cleaner shrimp’ in the current literature, with the potential to cause significant confusion. Indeed, this term has been used incorrectly for the shrimp Infraorder Stenopodidea, involving three families, Stenopodidae, Palaemonidae and Hippolytidae, and to represent all members of Lysmata and Stenopus. Caution is expressed in the use of grey literature and anecdotal observations to generate data on cleaning interactions, due to the presence of species complexes. Interest in cleaning organisms as biological controls in aquaculture is increasing due to their value as an alternative to various chemical ectoparasite controls. Reports of the importance of cleaner organisms in maintaining a healthy reef ecosystem has also been increasing and we review the current biological knowledge on cleaner organisms, highlighting areas that are understudied.     

Effects of supplementary fennel (Foeniculum vulgare) essential oil in diet on growth and reproductive performance of the ornamental fish,Convict cichlid (Cichlasoma nigrofasciatum)

This study evaluated the effects of diet containing 0 (C: control), 75 (D₁), 100 (D₂), 125 (D₃) and 150 (D₄) mg kg^?1 F. vulgare essential oil on growth and reproductive performance of C. nigrofasciatum. A total of 225 Convict cichlids female with mean weight (1.65 ± 0.02 g) were distributed into 15 glass aquaria (15 fish tank^?1) in triplicate treatments. During 40 days of experimental period, fish fed at 3% of their body weight daily. At the end of experiment, growth indices, gonadosomatic index and reproductive indices were determined. The best feed conversion ratio was obtained in D₄ (1.19 ± 0.03, P < 0.05). The addition of F. vulgare essential oil did not have any statistical effects on other growth indices (specific growth rate, weight gain and condition factor). Total survival rates in all treatments and control were uniformly high, ranging from 93% to 97% (P > 0.05). F. vulgare essential oil has no negative effect on survival rate of C. nigrofasciatum. There were no significant differences among protein content of fish body (P > 0.05). The lowest value of fat content (26.5 ± 0.5%) was observed in D_2, and it had significant difference with control and other groups (P < 0.05). The ash percentage in treatments fed with F. vulgare essential oil were higher than control fish (P < 0.05). The highest moisture content was detected in D₄ (72.68 ± 0.19%) and D₂ (71.23 ± 2%) groups. Among the used dosage, D₄ was the most effective dosage that could significantly increase GSI (11.06 ± 1.55%), fecundity (340 ± 21) and hatching ratio (92.33 ± 1.63%). There were no significant differences in diameter of eggs among various treatments (P > 0.05).     

The morphology–diet relationship and its role in the coexistence of two species of annual fishes

Annual fishes, which inhabit temporary pools with extremely limited habitat complexity and niche availability, display remarkable sexual dimorphism, rapid growth, and enormous investment into reproduction, all traits associated with high energy requirement. This study tests three hypotheses for two syntopic annual fishes (Austrolebias minuano and Cynopoecilus fulgens) found in six wetlands of southern Brazil: (i) considerable morphological differences result in low dietary overlap, (ii) sexual dimorphism in both species leads to intraspecific diet segregation, and (iii) dietary richness increases during ontogenetic development, and is narrower in C. fulgens than A. minuano due to morphological limitations imposed by reduced size. The diet of 82 A. minuano and 211 C. fulgens individuals was analyzed over two annual cycles. The morphology was characterized by 26 measurements covering the entire body of both species. There was no evidence of morphological specialization related to food competition and the diet of A. minuano and C. fulgens showed high overlap. High food availability, high predator abundance, and high connectivity of adjacent wetlands are likely the main mechanisms allowing coexistence of both species. Within species, sexual dimorphism did not result in a decrease in dietary overlap, which reinforces the idea that morphological differences between the sexes did not evolve as a mechanism to decrease food competition. Large A. minuano did not have a more diverse diet than the smaller C. fulgens; however, increase in body size allowed both species to ingest larger prey. Morphological variability in both species was mainly related to ontogenetic development and reproduction.     

The dietary zinc requirement of a benthic fish Paramisgurnus dabryanus

This study was conducted to assess the dietary zinc (Zn) on growth and antioxidant capacity of adult Paramisgurnus dabryanus. Zinc methionine (ZnM) of grade levels (0, 20, 40, 80, 120 and 160 mg/kg diet respectively) was supplemented, providing actual dietary Zn concentrations of 24.38, 28.03, 31.68, 38.98, 46.28 and 53.58 mg/kg diet respectively. P. dabryanus with an initial body weight of 5.21 ± 0.15 g were fed these Zn supplemented diets for 8 weeks. Results showed that the weight gain (WG) and specific growth rate (SGR) increased with increasing dietary Zn levels from 24.38 to 31.68 mg/kg, and then decreased above these levels. The hepatopancreas index (HIS) was the highest at 31.68 mg/kg, followed by 38.98 mg/kg. The enzymatic antioxidants in plasma and hepatopancreas firstly increased, and reached the peak at 31.68 or 38.98 mg/kg, then kept stable with the increase of dietary Zn levels. On the contrary, the content of MDA firstly decreased, and then increased. According to WG and T‐AOC in plasma, the Zn requirement was determined to be 32.02 and 32.24 mg/kg, respectively, based on regression analysis. The relatively low dietary Zn requirement of P. dabryanus may involve in the evolutional adaption of metal absorption and utilization to their habitat.     

The hidden companion of non‐native fishes in north‐east Atlantic waters

A tropicalization phenomenon of ichthyofauna has been described in the last decades in Galicia (north‐eastern Atlantic), with increasing reports of tropical and subtropical fishes appearing northward this distribution range. A search for parasites was carried out in the digestive tract of two specimens first captured in Galician waters: the prickly puffer Ephippion guttifer (Tetraodontidae) and the African stripped grunt Parapristipoma octolineatum (Haemulidae). Examination of E. guttifer showed high intensity of nematodes, from three different genera: Cucullanus (Cucullanidae), Hysterothylacium (Raphidascaridae) and Anisakis (Anisakidae), with demonstrated pathogenicity to humans. Molecular identification allowed the identification of Anisakis pegreffii, already described in the area, and first reports for European waters of Cucullanus dodsworthi, Hysterothylacium reliquens and a new Hysterothylacium sp. P. octolineatum showed a far lower level of parasitization, with two Hysterothylacium larvae, genetically identified as Hysterothylacium deardorffoverstreetorum, also its first report in the eastern Atlantic. Thus, possible ecological impact of the occurrence of two non‐native individual fishes in a new area could be remarkably higher if we see this issue through the lens of the parasitological perspective, as far as only two individual fish can harbour more of one hundred nematode parasites belonging to different species, most of them also new species for that area.     

Episodic predation of mammals by stream fishes in a boreal river basin

Stream‐dwelling fish typically feed on small prey items, such as benthic invertebrates, but maintain the capacity to opportunistically feed on rare, large‐bodied prey when available. However, consumption of particularly large prey is typically viewed as isolated events that are not organised spatially or temporally across watersheds. We assessed the occurrence of small mammals in the stomach contents of rainbow trout (Oncorhynchus mykiss) and Arctic grayling (Thymallus arcticus) over 13 years in the Wood River basin, Alaska (59°34′N, 158°48′W). Shrews (Sorex spp.) were the dominant mammals observed episodically every 2–3 years in the stomach contents of fish. Notably, shrew consumption was correlated in both Arctic grayling and rainbow trout within individual streams, and across the river basin in several subwatersheds. Predators of shrews were usually the largest individuals within each population, suggesting that smaller fish are gape‐limited and that dominant fish monopolised mammal prey. On average, 24% (11–38%) of Arctic grayling >298 mm (fork length) and rainbow trout >290 mm contained mammal prey during peak years. Small mammal populations often cycle every 2–5 years with well‐known functional and numerical effects for terrestrial predators, a dynamic that may be reflected in our 13 years data set of diet contents for aquatic predators. Although numerically infrequent when averaged over time, small mammal subsidies to streams may be episodically important to the energy budgets of long‐lived consumers in freshwater environments.     

First report of a cystic malformation on the upper jaw of hatchery‐reared allis shad Alosa alosa

The anadromous allis shad Alosa alosa has suffered dramatic population declines throughout Europe and is currently considered as endangered throughout its entire distribution range. In order to reestablish allis shad in the River Rhine, which formerly housed one of the largest and most important populations, an EU‐LIFE Project ‘The re‐introduction of allis shad in the Rhine system’ was started in 2007. In course of the LIFE+ Projects, allis shad larvae bred from genitor fish of the Gironde–Garonne–Dordogne population in France were reared in a pilot ex situ stock plant pilot facility in Aßlar, Germany. At an age of 1–2 months, about 100% of these fish developed approximately 0.5‐ to 0.8‐cm large, fluid‐filled, transparent cysts in conjunction with the upper jaw. The performed microbiological, virological, parasitological and histological examinations did not detect any infectious agents. Possible causative agents are discussed with regard to environmental factors and the nutrition of larvae. In conclusion, the observed malformations are considered a sign for a severe health problem and therefore a risk for the successful breeding of allis shad in aquaculture.     

Susceptibility of a number of Australian freshwater fishes to dwarf gourami iridovirus (Infectious spleen and kidney necrosis virus)

Megalocytiviruses cause high mortality diseases that have seriously impacted aquaculture, with the most frequent outbreaks occurring in East and South‐East Asia. The international trade of juvenile fish for food and ornamental aquaculture has aided the spread of these viruses, which have spread to Europe and Australia and other regions. Australian freshwater fishes were examined for susceptibility to infection with the exotic megalocytivirus, dwarf gourami iridovirus (DGIV), which belongs to a group with the type species, Infectious spleen and kidney necrosis virus (ISKNV). Fish were held at 23 ± 1 °C and challenged by intraperitoneal (IP) injection or by cohabitation with Murray cod, Maccullochella peelii (Mitchell) infected with DGIV. A species was deemed to be susceptible to DGIV based on evidence of viral replication, as determined by qPCR, and megalocytic inclusion bodies observed histologically. Horizontal transmission occurred between infected Murray cod and golden perch, Macquaria ambigua (Richardson), Macquarie perch, Macquaria australasica (Cuvier) and Murray cod. This indicated that DGIV shed from infected fish held at 23 °C can survive in fresh water and subsequently infect these naïve fish. Further, DGIV administered IP was highly pathogenic to golden perch, Macquarie perch and Murray cod. Compared to these species, the susceptibility of southern pygmy perch, Nannoperca australis (Gunther) was lower. Freshwater catfish (dewfish), Tandanus tandanus (Mitchell), were not susceptible under the experimental conditions based on the absence of clinical disease, mortality and virus replication. This study showed the potential risks associated with naïve and DGIV‐infected fish sharing a common water source.     

Stocking location and predation by marine fishes affect survival of hatchery‐reared Atlantic salmon smolts

Abstract Release strategies of hatchery‐reared Atlantic salmon, Salmo salar L., smolts were compared by studying survival and migration of smolts (n = 99) and their predators (Atlantic cod, Gadus morhua L., n = 8; and saithe, Pollachius virens (L.), n = 2) during the first 37 km of the marine migration using acoustic telemetry. Survivorship was higher in smolts released at the river mouth (30%) compared with smolts released in the river (12%). This was likely due to mortality or reduced migratory behaviour in fresh water. The marine mortality was 37% during the first 2 km after leaving the river (at least 25% mortality because of predation from marine fishes), and total marine mortality over 37 km was 68%. Detection‐depth data were useful for evaluating whether the tagged smolts were alive or predated; mortality during the first 2 km of outward migration would have been underestimated at 26% instead of 37% without the analysis of depth detection. Transmitters from consumed post‐smolts remained in predators for up to 47 days (average 29 days).     

Diet-induced phenotypic plasticity in the skull morphology of hatchery-reared Florida largemouth bass, Micropterus salmoides floridanus

Abstract –  Hatchery-reared Florida largemouth bass, Micropterus salmoides floridanus , feed on inert pellet food while their wild counterparts capture elusive prey. Differences in levels of prey elusivity often mandate the use of alternate methods of prey capture. This study examines whether elusivity-based variation in prey capture translates to a phenotypic change during skull development, and if this change results in a functional difference in the feeding mechanism. The developmental pattern of the skull was conserved between hatchery and wild bass until 80–99 mm TL. At this point, wild bass quickly developed morphological changes of the jaw apparatus including a more fusiform head and elongated jaw structures. Natural development in hatchery bass, however, was retarded at this size. Post-release, the skulls of hatchery fish converged towards those of wild bass by 135 mm TL. Despite variation in skull development, no theoretical advantage in food capture was found between these two groups.     

Practical diets with essential oils of plants activate the complement system and alter the intestinal morphology of Nile tilapia

The effect of the essential oils (EOs) of peppermint, Mentha piperita L., and tea tree, Melaleuca alternifolia (Maiden & Betche) Cheel, on the haematological, biochemical, and immunological parameters and intestinal morphology of Nile tilapia, Oreochromis niloticus L., was evaluated. Fish (58.09 ± 5.87 g) were fed 100 mg/kg and 250 mg/kg of each EO and sampled on days 7, 14, 30 and 60 after starting supplementation. The haematological and biochemical parameters were not altered by the supplementation of EOs compared to the control (p > .05). With regard to the immunological parameters, the activation of the complement system of fish fed 250 mg/kg peppermint and 100 mg/kg and 250 mg/kg tea tree EOs were significantly higher compared to the control after 60 days of feeding (p < .05). The complement system plays an essential role in innate immunity and contributes significantly to the acquired immune response; thus, its activation through supplementation with EOs is promising for the formulation of nutritional additives in aquaculture. Regarding intestinal morphology, fish fed 250 mg/kg tea tree EO presented higher villus size compared to all other groups (p < .05), which represents a healthier gut. These fish present a larger intestinal surface, which can result in better absorption and utilization of the nutrients. Based on the responses found in this study, both EOs were considered promising for the formulation of feed additives for Nile tilapia.     

Effects of dietary supplementation with β‐glucan and synbiotics on growth,haemolymph chemistry,and intestinal microbiota and morphology in the Pacific white shrimp

The goal of this study was to investigate the effects of dietary supplementation with β‐glucan and microencapsulated probiotics (Bacillus subtilis or Pediococcus acidilactici) on growth performance, body composition, haemolymph constituents, and intestinal morphology and microbiota of the Pacific white shrimp Litopenaeus vannamei. Four treatment diets [basal diet (C), β‐glucan‐containing diet (β‐glu), β‐glucan plus B. subtilis‐containing diet (β‐glu+Bs), and β‐glucan plus P. acidilactici‐containing diet (β‐glu+Pa)] were fed to L. vannamei for 90 days. Shrimp fed the β‐glu and β‐glu+Pa diets exhibited similar growth performance and body protein content, which were significantly higher than those of shrimp fed the control diet (P < 0.05). No significant differences in haemolymph triglyceride, cholesterol, protein, haemolymph urea nitrogen or chloride were detected among the experimental diets. However, dietary β‐glucan alone increased the haemolymph glucose level and osmolarity (P < 0.05). Synbiotic supplementation had greater effects on intestinal microbiota and morphology than dietary β‐glucan alone. For example, β‐glu+Bs increased the number of intestinal lactic acid bacteria and decreased the number of Vibrio spp. (P < 0.05), and β‐glu+Pa increased the height of intestinal villi.     

Time kinetics of fatty acid changes in phospholipids following enrichment and starvation of Artemia franciscana with a main focus on docosahexaenoic acid

The main objective was to study time kinetics of change in important highly unsaturated fatty acids (HUFAs) in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of Artemia franciscana nauplii and juveniles following enrichment and subsequent starvation. Samples of Artemia nauplii were taken at variable times (0.5–24 h) following enrichment and starvation. Samples of Artemia juveniles were taken after 2, 3 and 4 days of cultivation. No docosahexaenoic acid (DHA) was found in PC and PE of Artemia nauplii during the first hour of enrichment, while a significant (P < 0.05) increase was found in total lipids (TLs). The content of DHA in PC and PE increased thereafter steadily from 1 to 8 h of enrichment. DHA in PC and PE during enrichment (1–8 h) and following starvation (8–24 h), respectively, increased and decreased significantly (P < 0.05), but at a lower rate than that in TL. Moreover, juvenile Artemia (2–4 days) contained a relatively low level of DHA in TL compared with enriched Artemia nauplii, but the content of DHA in PC and PE was similar. The results open perspectives for both industry and science. For scientific studies, the lag phase in HUFA enrichment makes it possible to produce Artemia nauplii with variable relative HUFA enrichments in phospholipids and TL.     

Effect of guava Psidium guajava (L.) aqueous extract diet on growth performance,intestinal morphology,immune response and survival of Oreochromis niloticus challenged with Aeromonas hydrophila

An 84‐day feeding trial was carried out to evaluate the effects of aqueous Psidium guajava leaf extract (PGE) on growth, intestinal morphology, physiology, immune response and susceptibility of Oreochromis niloticus to Aeromonas hydrophila. Diets containing 0% (P0), 0.25% (P1), 0.50% (P2), 0.75% (P3) and 1.00% (P4) PGE were fed to triplicate groups of fish (mean weight; 1.32 ± 0.04 g) for 84 days. After the 84‐day feeding trial, test fish were injected with pathogenic A. hydrophila and then fed for 14 days. More feed were consumed in groups of fish fed PGE‐treated diets and resulted in significantly higher weight gain and feed intake. Incidentally, there was an increase in the calculated area of absorption of fish fed PGE diets, as accounted for by marginally higher villi length and width. Antioxidant and immune response were improved with PGE inclusion in diets as total protein, superoxide dismutase, glutathione peroxidase and glutathione S‐transferase significantly increased (p < 0.05) in fish fed PGE diets. Results of the challenge test with A. hydrophila revealed that the highest mortality (100%) was recorded in P0. This study revealed that inclusion of P. guajava extract in the diet of O. niloticus improved growth, nutrient utilization, immune system and survival of O. niloticus fingerlings.     

Effects of fish meal replacement by soybean meal with supplementation of functional compound additives on intestinal morphology and microbiome of Japanese seabass (Lateolabrax japonicus)

A 15‐week trial was carried out to estimate the effects of functional compound additives (FCA) on intestinal morphology and microbiome in Japanese seabass, Lateolabrax japonicus, fed diets with soybean meal (SBM) partially replacing fish meal (FM). The formulation of FCA was the mixture of antioxidant, immunopotentiator and Mintrex^® trace elements. Four isonitrogenous (45%) and isolipidic (11%) diets, including FM42 (the control group without FCA), FM35 (15% FM protein substitution level with FCA), FM21 (50% FM protein substitution level with FCA) and FM21‐N (50% FM protein substitution level without FCA) were formulated. Quadruplicate groups of seabass (initial average weight 125.65 ± 0.60 g) were randomly handfed each diet twice daily in seawater floating cage. No significant differences were found in microvillus height (HMV) and muscular thickness (MT) of distal intestine among fish fed FM42, FM35 and FM21, whereas the HMV, MT and fold height (HF) in seabass fed FM42 were significantly higher than those of seabass fed FM21‐N (P < 0.05). Seabass fed FM21‐N had more obvious enteritis parameters such as tissue disruption, wider lamina propria and less mucosal fold than those in seabass fed FM21. High‐throughput sequencing technology of gut flora showed that Proteobacteria was the most dominant bacteria at phylum level among groups. There was no significant difference in relative abundance and composition of gut microflora among groups. In conclusion, supplementation of FCA might partially eliminate SBM‐induced enteritis, but the composition and relative abundance of intestinal microflora were not affected by dietary treatments.     

Inbreeding and disease in tropical shrimp aquaculture: a reappraisal and caution

The disease crisis facing shrimp aquaculture may be propelled, in part, by an interaction between management practices that cause inbreeding, and the amplification by inbreeding of susceptibility to disease and environmental stresses. The study describes and numerically simulates gene flow from Penaeus (Litopenaeus) vannamei hatcheries that employ a ‘Breeder Lock’ to discourage use of their PL as breeders, through ‘copy hatcheries’ that breed the locked PL, to inbred shrimp in farm ponds. Re‐analysis of published data shows that inbreeding depression under stress is exceptionally strong in shrimp. Inbreeding is currently overlooked as a problem because: (1) procedures recommended for well‐managed hatcheries do not consider their implications for the copy hatcheries that supply most farmed shrimp (estimated 70%), (2) inbreeding in hatcheries is often reported as zero even though zero is the mathematical expectation of the usual estimator (Fis, fixation index) whatever the true genealogy of the broodstock. Simulation shows, however, that inbreeding can be estimated with Wang's trioML estimator, that Fis can differentiate Breeder Locked from copy PL and that simple tests can verify the lock status of PL. The importance of inbreeding should be re‐evaluated in the context of disease and environmental stress. Unrecognized inbreeding may increase the incidence, prevalence and lethality of WSSV, IHHNV, EMS (AHPND) and other diseases.