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These mesopelagic fishes are the dominant species in the ocean and are the vertebrates with the largest biomass on the planet10. The total biomass of these species has been estimated at approximately 1000 million tons11, but these values may be underestimated by one order of magnitude12. Moreover, the estimated contribution of these species to deep water respiration would be approximately 10%12, and thus, their role in ocean ecosystems and their contribution to ocean biogeochemical cycles have vital importance.
Myctophids play an important role in energy transfer in pelagic ecosystems, linking the planktonic organisms such as copepods, ostracods and larvaceans17,18, with pelagic fish19, cephalopods20, seabirds21 and marine mammals22. Despite the intense predation they support, lanternfishes are highly abundant23, and it is important to understand their population dynamics, particularly their reproductive biology12. Moreover, as a potential fishery resource24, interest in the biology, ecology and population dynamics of these mesopelagic fishes is progressively increasing25,26.
Sex was estimated from 518 samples due to external body dimorphism. Males present a large supra-caudal gland that allows them to be differentiated externally from females. The sex was confirmed after dissecting the fish and observing the gonads macroscopically. However, the state of maturity was determined for only 459 individuals through gonad macroscopic observation, following the classification criteria for fishes53 to classify these stages as immature, developing, spawning capable, regressing and regenerating. The macroscopic scale of maturity was validated with histological analysis, including all maturity stages, considering the standard terminology for describing reproductive development in fishes53. A random stratified sampling strategy was applied. For each macroscopic ovary stage, 10 gonads each were selected and processed histologically for each ovary maturity development stage.
Another method used to determine the spawning season is based on the temporal variation in the frequency of individuals in each stage of maturity, which was determined by macroscopic observation of the gonads. To do this, the number of fish in each stage of maturity and their frequencies were calculated. To estimate GSI at 50% maturity (GSI50), a logistic function was fitted to the fraction of mature fish per 0.5 GSI interval, for males and females, using a nonlinear least-squares regression. The logistic equation was:
A total of 50 ovaries were fixed and preserved in 4% buffered formaldehyde for histological analysis to verify the previously assigned macroscopic maturity stages. For this, the fixed tissues were dehydrated in a series of ethanol solutions, cleared in isoparaffin H, and then embedded in paraffin in a vacuum chamber. Slices of tissue were sectioned at 4 m and stained with Harris haematoxylin followed by eosin counterstaining55. To describe the scale of gonadal maturity, the standardized nomenclature53 was used. The size of the oocytes within the four ovarian developmental stages were determined to describe the type (synchronous or asynchronous) of maturation and spawning based on ovaries that were previously processed histologically. The oocytes were removed from the ovary then placed in a vial and pipetted vigorously or full separation. The contents of the vial were then poured into a counting chamber, and the isolated oocytes were measured. The first 100 oocyte diameters were measured to the nearest 0.01 mm and classified based on histological correspondence images.
Finally, 84 samples of female gonads were collected to estimate the batch fecundity (BF)56,57 through the gravimetric method, which was calculated for each female as the number of oocytes per unit weight multiplied by the total ovarian weight40.The proportion of the subsample from which the oocytes were extracted was evaluated with a target coefficient of variation (CV) of oocytes per unit weight of less than 5%58. The oocytes were manually released from the ovarian stroma and then counted using a stereoscopic microscope. Ovaries that did not contain early stage postovulatory follicle complex (POFs) were used because the presence of these indicate that some eggs have been already released59. In these ovaries, the oocytes at the most advanced stages, primarily Vtg3 and hydrated oocytes (H), were counted to estimate the batch fecundity56,57.
The statistical analysis was performed using the R programming language (V.3.6.0)60. Batch fecundity data from other studies are also shown for comparison purpose40,61. We obtained the values from the data points of figures using GetData Graph Digitizer V.2.26. The sampling map was generated using the geographic information system QGIS Development Team (V.3.12.1)62.
All necessary permits for sampling and observational field studies have been obtained by the authors from the competent authorities and are mentioned in the acknowledgements, if applicable. The study is compliant with CBD and Nagoya protocols.
The length frequency distribution showed the presence of two groups of lengths during the sampling period. From May to December, individuals of relatively small size (smaller than 60 mm SL) predominated, while larger fish (larger than 65 mm SL) were more abundant from January to April (Fig. 2).
Variation of relative condition factor (K) for each four-months period throughout the available range of lengths available for N. resplendens.(Red dots: Females individuals; Blue dots: males individuals).
Relationship between standard length (SL, mm) and gonadosomatic index (GSI) of N. resplendens by each four-month periods. Horizontal dashed lines indicate the GSI at 50% sexual maturity (GSI50), and arrowheads on the horizontal axis indicate the minimum size at sexual maturity for each sex.
The description of four different stages of development in the female gonads was performed, and the macroscopic observations were validated with gonad tissue histology. The standardize nomenclature53 was used to describe the ovarian development of N. resplendens in four reproductive phases described below (Fig. 8):
The analysis of the inner structure of oocytes in N. resplendence showed that Vtg1 oocytes were characterized by the presence of small vacuoles, the phases Vtg2 and Vtg3 were defined by the presence of vacuoles in the central and perinuclear areas of the oocyte, and of small yolk granules at its margin (Fig. 8).
The proportion of males and females in the entire sample was a 1:1 ratio, but we did not find any significant differences in their proportionality according to season. In this regard, the greater number of females observed in January to April could help maximize the egg-producing biomass34,65, and the opposite tendency recorded during May to August, when males predominated, could enhance the likelihood of mating but at the expense of a decreased number of egg producers in the population66. These differences in the sex ratio during the year (or years) and even with depth intervals have been observed in several species of myctophids, such as Benthosema pterotum67.
The ratio of males to females decreased with SL, and most individuals in the largest class were females, as observed in many other myctophids37,39,40. This variation in the sex ratio with length might be related to faster growth and/or a longer life span of females in relation to males65. This has been documented in several myctophid species belonging to the Notoscopelus genus, where females grow faster than males and reach a larger maximum size46,68,69.
Previous studies have found that males N. resplendens begin to develop the large supra-caudal gland at 37.5 mm SL (41 mm TL), reaching sexual maturity at 59.65 mm SL (66 mm TL)70. Our data indicate that in the Canary Islands region this gland begins to develop at a smaller length than that reported by Hulley13 (25.29 mm SL, i.e., 27.14 mm TL), and consequently, the average length of first maturity was also lower (56.61 mm SL, i.e., 61.60 mm TL) than previously estimated by the abovementioned author. Sexual dimorphism in luminous organs is known in many myctophids71,72. These caudal organs are considered to be related to sexual recognition in myctophids because they can produce volleys of very fast and high intensity flashes72,73. Studies on B. pterotum61 suggest that this bioluminescent sexual signalling might be used to facilitate communication between sexes at night, and this is possibly related to more efficient mating.
In contrast, females of N. resplendens were mature at a larger size than males, with a length at first maturity of 60.34 mm SL. In other myctophid species, males mature at smaller sizes than females29,30,34,65. This would contribute to the greater biomass of mature females than males and to maximizing the egg-producing biomass.
In other studies of myctophid species, for example, in the Diaphus and Benthosema genera, the range of size at maturation is between 24.5 and 120 mm SL38,40,61, depending on the growth rate of the species and the region in which it is found. This could be the reason we found variability in the size of first maturity in the same species.
Therefore, the lower K values observed in specimens of N. resplendens caught in March and May are related to the end of the reproductive season in the waters of the Canary Islands, during a similar period to that observed in the neighbouring waters of Mauritania, where spawning takes place in winter and spring77. Moreover, N. resplendens showed reproductive activity during winter and early spring (January to April), when the percentage of mature individuals was 70.2%, with reproductive activity occurring particularly in the larger length range.
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