Merluccius merluccius) as a case study. Our work evidenced sophisticated seasonal oocyte recruitment dynamics and patterns, mostly driven by a low-cost predefinition of fecundity as a function of fish body size, likely influenced also by environmental cues. Fecundity seems to be defined at a much earlier stage of oocyte development than previously thought, implying a quasi-determinate – rather than indeterminate – fecundity type in hake. These results imply a major change in the conceptual approach to reproductive strategies in teleosts. These findings not only question the current binary classification of fecundity as either determinate or indeterminate, but also suggest that current practices regarding potential fecundity estimation in fishes should be complemented with studies on primary oocyte dynamics. Accordingly, the methodology and approach adopted in this study may be profitably applied for unravelling some of the complexities associated with oocyte recruitment and thereby SRP variability.Information on temporal variations in stock reproductive potential (SRP) is essential in fisheries management. Despite this relevance, fundamental understanding of egg production variability remains largely unclear due to difficulties in tracking the underlying complex fluctuations in early oocyte recruitment that determines fecundity. We applied advanced oocyte packing density theory to get in-depth, quantitative insights across oocyte stages and seasons, selecting the commercially valuable European hake ( In fish populations, recruitment results from a myriad of interactions, beginning with factors that determine the level of egg production3 which, in turn, can be traced back to fundamental processes influencing oogenesis5. Related parameters – such as fecundity and length of spawning season – are therefore key in defining stock reproductive potential (SRP)6 and, consequently, need to be taken into consideration in population dynamics studies7. However, properly quantifying the formation of the smallest oocytes (primary oocytes) and thereby better understanding why fecundity varies is a highly complex issue requiring advanced methodology. An added complication in such studies is that, in order to track the fate of the sex cells, the ovarian samples need to be collected over a sufficiently long time-scale to cover the different parts of the reproductive cycle. Furthermore, the impact of environmental stressors and cues on oogenesis can change markedly throughout the year.In modern natural resource management, maintaining a population’s reproductive potential above a certain minimum threshold value  is one of the most important practical components of sustainability plans for populations and ecosystems services8. The temporal relation between oocyte recruitment from primary to secondary growth and the spawning season defines the fecundity type, which ranges from clearly determinate to indeterminate9. In species with determinate fecundity, oocyte recruitment is completed before the onset of the spawning season. This means that potential annual fecundity can, in principle, be estimated by the standing stock of prespawning, secondary growth oocytes since, in the beginning of this phase of development, those oocytes, which will subsequently be released during the spawning season, are stored to be later matured in lots (cohorts). In contrast, indeterminate species are capable of recruiting oocytes to secondary growth throughout the spawning season. Thus, direct estimation of potential annual fecundity is not possible because the total number of oocytes produced per season is not fixed prior to spawning. Instead, annual fecundity is estimated by multiplying typical batch fecundity (number of eggs spawned in a single spawning event) with the number of batches released10. Therefore, the appropriate method for estimating egg production depends on the fecundity type of the species in question. However, despite its importance in the reproductive biology of fisheries, the fecundity type – determinate or indeterminate – remains unknown for many species11. In this regard it seems that, rather than simply obtaining “snapshots” of secondary growth oocyte development, dedicated studies of oogenesis are needed12. Moreover, the question as to whether the fecundity type is genetically predefined, or modulated by habitat and environmental characteristics as an ecophenotypic response, is mostly unresolved13, which calls into doubt the rigid labelling of species into one type or the other, which is traditionally the case in most marine laboratories. In fact, several studies have already linked fecundity type to geographic distribution14, spawning season and the energy allocation strategy during reproduction15, suggesting varying degrees of plasticity.The course of oogenesis goes through three main steps: proliferation of oogonia in the lamellar germinal epithelium followed by development of primary oocytes and then secondary oocytes (which ultimately includes ovulation)11. The advanced Oocyte Packing Density (OPD) theory16, which builds on the digital auto-diametric method17 with facets of stereology and volume-based theory, provides a far better understanding of oocyte recruitment, as even the smallest oocytes in different stages can be reliably quantified independently of fecundity type20. Although the use of OPD theory is certainly gaining momentum, it is rightly pointed out that its formulations require special attention by new experimenters if they are to be executed correctly. Such efforts should, however, be balanced against the fact that existing studies on indeterminate species remain scarce, typically providing a limited view of this highly complex oocyte recruitment process, and focusing, for example, mostly on the spawning season, even though teleost reproductive cycles are well known to show circannual periodicity. So, the underlying seasonal oocyte production has, thus far, rarely been adequately addressed quantitatively18.In response to all of these uncertainties regarding basic and applied aspects of identifying fecundity patterns, theoretical and methodological advances have been made over the last decade or so, leading to far more detailed and accurate fecundity studies and also, in several cases, using less labour-intensive methods21. Amongst the long list of external environmental variables possibly involved, photoperiod has been described as one of the most common cues, triggering gonad development in temperate fish species, while factors such as temperature, food availability and physiological status undoubtedly act as drivers, regulating the rate of oocyte recruitment and development22. However, in indeterminate species with a protracted spawning season, it quickly becomes extremely complicated to ascertain and isolate the varied roles of these external cues and drivers due to the asynchronies in oocyte development patterns23, and the fact that such lengthy experiments are difficult to run under realistic conditions in the laboratory. All things considered, there is a clear need to improve our understanding of the underlying regulation of oocyte recruitment and fecundity pattern, especially in species with an apparent indeterminate fecundity. This requires not only extensive field sampling covering the full reproductive cycle and access to detailed environmental information, but also advanced laboratory routines to track stage-specific oocyte development in reliable, quantitative terms. In this article, we address the fundamental problems of such research and the associated methodologies, in the course of a complex research effort of this kind on the European hake (Merluccius merluccius) from the Galician shelf. This species has a highly complicated reproductive strategy but, in general terms, it is among the most data rich species in the world due to its prominent role in the fishing industry as one of the main commercial target species of the French, Spanish and Portuguese fishing fleets in the North Atlantic. A protracted spawning season has been documented including several spawning peaks or seasons within the year24. Generally, the species is considered a batch spawner, with an asynchronous oocyte development and continuous indeterminate fecundity18. For the southern stock, a high degree of variability in several reproductive traits has been reported25; however, certain fundamental aspects of its reproduction are still barely touched upon26. Altogether, the specific aims of the research described in the present article were (i) to quantify the seasonal dynamics of the whole range of oocyte stages in European hake, applying OPD theory – with special emphasis on early oocyte recruitment, which is widely recognized as a “black box” in the teleost literature; (ii) to re-examine the traditional classification that classifies hake as a species with an indeterminate fecundity pattern; and finally, (iii) to identify potential links between oocyte recruitment and environmental cues, while bearing in mind that multiple cues might be involved.It seems important, therefore, to gain a better understanding of the process of recruitment of the oocytes to be developed during the spawning season, as well as of the factors underlying changes in egg production, both of which are linked to stock productivity27 and herring28. This included the appearance and formation, in the cytoplasm, of the so-called circumnuclear ring (CNR) – which, being rich in organelles and RNA, is homologous with the Balbiani body and, therefore, a central criterion for staging these oocytes  manifested markedly different patterns as a function of ovarian growth – or more specifically, the ovarian phase defined by the presence of the most advanced oocyte stage (MAO)  at earlier ovarian phases, but then steadily declined towards spawning , starting off with a sharp increase when MAO approached secondary growth  were observed for subsequent secondary growth oocytes, i.e. in the stages defined by the presence of cortical alveoli (CAO), vitellogenic (EVO and VTO), then migrating nucleus (MNO) and finally, hydrating oocytes (HYO)  showed a pattern similar to PVO4a and PVO4b oocytes  Fig. , showed ing Fig. . In conting Fig. , showed wth Fig. . SimilarYO) Fig. . While PO4a Fig.  and fromO4a Fig. , referrites Fig. . Ovariesabsolute number of oocytes in different stages in the whole ovary, found by multiplying OPDi with the corresponding ovary weight adjusted for TL, i.e. relative TL-based NOi, was recognized as an effective way forward to pinpoint oocyte production, since individual body size differences : PVO2 increased noticeably in number (peaking at ≈2500 oocytes cm-3), while PVO1 and PVO3 increased only modestly. Meanwhile, as seen in Fig. −3, respectively), although this is low compared to the same values for PVO stages 1 to 3 (≈1300 oocytes cm−3). The general decline in number continued as the PVOs developed and thereby grew in cell size  and, as shown in Fig. −3). The seasonal variation of PVO4c and CAO abundance was roughly similar, with generally higher numbers during winter and spring, more so in the case of PVO4c (cf. ≈180 and 130 oocytes cm−3 before and after summer solstice).As seen in Fig. −3, i.e. 30% lower than for CAO. For both vitellogenic stages, the lowest numbers were found in July (≈25 oocytes cm−3), but there also three concurrent, but diminishing peaks throughout the year. This pattern of oocytic abundance appears to be linked with “pulses” of higher oceanic temperatures and subsequently more-concentrated spawning activity  data on postovulatory follicles (POFs) presented three peaks (or possibly four), the first, in January, being the most pronounced, followed by a second smaller peak and a third which was intermediate in size. Finally, in Fig. i levels of atresia , late alpha (Lα) and β stages of atresia), similar patterns can be seen, most noticeably with the Lα stage, although the peaks are delayed compared to the VTO peaks. During the third spawning peak, coinciding with the warmest waters  and medium or late vitellogenesis (VTO) Fig. , typical−3), there was a marked increase (by ≈1750 oocytes cm−3). The PVO4b pattern begins to resemble those of the PVO4c to VTO stages, shown in Fig. Before addressing this topic, it should be noted that data on PVO stages 1, 2 and 3 were pooled, for simplicity, as fluctuations during each of these stages were very similar, with only PVO2 showing a certain degree of deviation Figs.  and 3A. During the developing and spawning capable ovarian phases, larger females generally had the highest numbers of oocytes Table . For insTo further address fecundity dynamics in European hake, the total number of egg batches a representative individual (within each 10-cm category) could possibly produce over the three spawning seasons in question SS1 to 3) were estimated theoretically. In the case of 60–70 cm females, this number was 66 , provided all oocytes from PVO4b onwards ended up as eggs . Interestingly, the total amount of PVO1-3, which are not contributing to annual fecundity, also increased significantly with female size,  and SS1 showed significant lower values  , reflect the potential fecundity. A 60–70 cm female, for example, would then produce ≈1.8 million oocytes, spawned in 6 egg batches over 0.8 months in SS1; ≈1.5 million oocytes in 8 egg batches over 1.1 months in SS2; and 1.3 million oocytes in 8 egg batches over 1.1 months in SS3 (Table 05) Fig. .Figure 530. Here we have shown that oocytes are recruited in hake much earlier than previously thought22. This result challenges the classic definitions of determinate and indeterminate tactics31 and hence our understanding of fish reproductive strategies. In our opinion, this finding is not exclusive for hake and very likely happens in many, if not most, of the oviparous and pelagophil species. We found that the egg production is mostly dependent on female size, and thereby on female energetics, i.e. their feeding capacity13. However, the spawning dynamics differ not only by female size, but also among seasons. The methodology and approach taken in this study appear well suited to throw light on the complexities associated with fish reproductive productivity.Oocyte recruitment is influenced by a complex interaction of temporal events and maternal features which, in the case of marine teleosts, have generally received little attention from researchers, and a number of assumptions have therefore been made regarding their reproductive strategiesi). This enabled us to make a highly precise exploration of the temporal dynamics of oocyte recruitment, accounting also for the formation of the very smallest primary oocytes, three classes of atresia, post-ovulatory follicles (POFs) as well as the level of other tissue components, including blood capillaries. All in all, the present methods, securely founded on earlier works arising from the introduction of the OPD theory nearly a decade ago16, should provide realistic approximations of the seasonal, numerical oocyte production (NO), which is particularly relevant in species with complex reproductive strategies. The results of a previous methodological study on early oocyte ( > 120 µm) recruitment in European hake18 cannot be directly compared because a much more detailed staging key for PVOs was used in this study, covering the full yearly cycle instead a part of it. However, in-built validation tests showed a close correspondence between estimated  and observed values , the latter having been calculated from whole-mounts29. The results indicate that each of these two vitellogenic cohorts will subsequently develop into single batches, thus supporting the use of (NOEVTO + NOVTO)/2 as a proxy for batch fecundity.State-of-the-art laboratory techniques  were used to estimate stage-specific oocyte packing density  the appropriateness of the current oocyte staging scheme, originally applied to cod32, a determinate spawner with a group-synchronous oocyte development. Here the dynamics of these oocyte stages are evaluated in a teleost displaying an asynchronous oocyte development. Based on our results, sexually immature specimens may be characterized by the presence of PVO3 and PVO4a as the most advanced oocytes (MAO) and small amounts of blood, while ovaries showing oocytes in PVO4b and 4c stages as MAO correspond either to regenerating specimens or specimens at the onset of sexual maturation, i.e. in puberty. This view is supported by previously published results on cod and herring32. Consequently, we argue that the oocyte stage PVO4b, or the final formation of the circumnuclear ring (CNR) represented by PVO4c, could be incorporated in future studies as an early marker of sexual maturation schedules in European hake, and likely in many other teleosts as well. Note, however, that identifying regenerating individuals from those in puberty requires a careful histological check of the presence/absence of actual spawning markers (e.g. POFs)33.Oocyte development is a continuous process, which, logically, makes it difficult to always confidently classify stages. In contrast, and although the annual mean abundance was similar to that of PVO4a, the PVO4b dynamics were much more – although not completely – related to spawning activity. Furthermore, the PVO4b abundance appeared highest in the first half of the year (after the winter solstice). A second important drop in oocyte numbers was found from PVO4a-b to PVO4c, while from PVO4c to VTO this reduction was more modest and occurred more steadily. As distinct from the postulated long-term reservoir of PVO1-3, these oocytes were obviously being recruited for subsequent spawning, since the abundance of PVO4c onwards, showed three peaks . Hence, the remaining issue is to elucidate the role of the intermediate or transitional primary stages, PVO4a and PVO4b, which probably have a mixed role as a reservoir and “recruitment spot”. Importantly, their abundance was low in immatures, in contrast to PVO1-3, (note here our above comment that “immatures” might be split between those being true “immatures” and those in early puberty), equally higher in developing and spawning capable females, but, in the case of PVO4b, decreased significantly in regressing and regenerating phases, although far from reaching the zero values observed for vitellogenic oocytes. Hence, our suggestion is that PVO4a should be considered a medium-term reservoir for the current spawning season or for the following one(s); they may or may not be recruited, depending on external and internal factors (see next section). In stark contrast, PVO4b seems closely associated with oocyte recruitment within the current period. Having said that, a precise timing of when and how oocyte recruitment to PVO4b ends was not established in this study, although we did see clear peaks in PVO4b in spawning capable females. In any case, the PVO4b stage should be considered as a pool of cells that can, potentially, be spawned during the up-coming season, in line with the expected realized fecundity and thereby the duration of spawning, as discussed later. The fate of the observed surplus of PVO4a-b at the end of the spawning season remains speculative: these cells might be either broken down through apoptosis or atretic processes34, or revitalized as part of next spawning season’s production (see below).The major reduction in oocyte numbers between PVO1-3 and PVO4a probably means that there is an actual “reservoir” of the smallest primary oocytes. In other words, this high number of PVO1-3 implies that not all of them will be recruited to be spawned during the current season. Moreover, their temporal, numerical (NO) trend, being opposite to the one seen for vitellogenic oocytes, reinforces the idea that they are not specifically dedicated to the up-coming spawning season(s) but to future ones. We found that this PVO1-3 reservoir is generally built up during the second half of the year, but also that this reservoir is actively utilized in the early transformation from immature to developing ovarian phases. However, the fact that this reservoir is so large complicates any evaluation of direct dynamic links to subsequent oocyte stages. Despite seeing a three-fold decrease in NO values from PVO3 to PVO4a, these two stages followed a similar seasonal pattern independent of spawning activityet al.5  clarify that the level of fecundity in herring seems predefined very early on in oogenesis, apparently in response to much earlier feeding opportunities. In our staging system, the PVO4 stages are specifically classified based on the development of the circumnuclear ring (CNR) in cytoplasm, but only the PVO4b stage is characterized by a well-formed and evident CNR. For herring, the appearance of the CNR is likely triggered around the winter solstice, considered to be “the first decision window”, while the spring equinox in this species may operate as “the second decision window”28. In fact, photoperiod has been linked to initiation of early oocyte recruitment or development, at least in some high-latitude teleosts where day length, and thereby the strength of this photic signal as an environmental cue, changes markedly throughout the year35. Here we could not establish any robust relationship between day length and oocyte recruitment, but we did observe a concurrence between the increase in PVO4a and b abundance and the increase in daylight hours after the winter solstice. Consequently, their observed lower numbers during the second half of the year might simply be explained by the subsequent transfer to later developmental stages. However, hake is considered an income breeder36, i.e. the acquired energy is immediately used for reproductive investment, meaning there is no need for related, extensive storage37. In other words, environmental factors triggering oocyte production (at least vitellogenesis) must necessarily be related with instantaneous energy demands. So, taken together, it seems reasonable to believe that photoperiod may play a potential role in triggering the mobilization of oocytes from “reservoir to recruitment” while the rate at which oocytes develop may be regulated by other environmental factors, e.g. upwelling events attracting foraging fish (the main hake prey)38, and the temperature regime experienced40.It seems clear, therefore, that the oocytes to be spawned in each spawning season of European hake are “labelled” at some point during the PVO4b stage, but, of course, all these cells will not necessarily make it to the end of the reproductive cycle due to varying incidences of apoptosis or atresia. In this line, the statistical findings of dos Santos Schmidt 4. However, it is also affirmed that the preceding primary growth (PG) phase is primarily gonadotropin-independent42, and the production of CAO, the “bridge” between primary growth (PG) and secondary growth (SG), might possibly be related to other hormonal pathways as well43. Our results indicate that a spawning-related dynamic is already in place at the PVO4b stage, i.e. well before gonadotropin-dependent regulation. This is a striking finding because it implies that oocyte recruitment  takes place much earlier than traditionally thought. Given the similarity of the oogenesis among fish species30, this result has a broad implication in fish reproduction research. Whether this implies that the present focus in the fish fecundity literature on SG rather than PG processes should be reconsidered is a matter of discussion, but given that oocyte recruitment is principally determined during PG, as evident in European hake, the standard definition of determinate and indeterminate fecundity should be, at the very least, revisited. This is because both concepts today relate to oocyte recruitment patterns during SG, or more specifically, during the spawning season as such.Although a series of gaps exist in our knowledge regarding oogenesis in teleosts, it has been firmly established that oocyte development is stimulated by gonadotropins44. The observed three peaks or seasons involved different level of spawning activity , which seems to happen concurrently with upwelling events; egg release during upwelling likely promotes successful reproduction in these types of ecosystems45. Also, a protracted spawning season is tightly coupled with an income breeding strategy and indeterminate fecundity46; in spawning hake, this is evidenced by intense feeding47 and secondary growth oocyte dynamics48, respectively. However, our findings challenge today’s thinking: assuming the standing stock of oocytes at PVO4b-VTO stages represents the potential fecundity, the estimations undertaken here give a highly plausible realized fecundity, as would be the case in a determinate fecundity species. Moreover, while we would expect such figures to change between spawning seasons in an indeterminate/income breeder species in accordance with food availability, they actually change with fish size, suggesting strong maternal effects44. Note here that production of the smallest oocytes, logically, requires little energy.The southern stock of European hake shows a very protracted spawning season, virtually covering the whole year, but with several peaks, as documented here and earlier49. Intense atretic activity was observed both in this and earlier studies on hake50, but only after the spawning season ceases, with the possible exception of summer time where atresia coincided with the spawning peak. This picture has been linked to an indeterminate strategy30, or a “mopping-up” process51. Altogether, this supports the impression that down-regulation of fecundity in hake occurs as a consequence of the developmental rate of each cohort (defining the batch fecundity and spawning frequency), which, hypothetically, could be mediated by environmental factors, and which results in the presence of remnants of underdeveloped oocytes to be removed by apoptosis or atresia once the spawning is over.Down-regulation of the more advanced oocytes is often seen as a process of matching the available energy52. This also applies to the genus Merluccius, which displays large phenotypic plasticity in traits such as fecundity and spawning length across stocks inhabiting waters located at different latitudes, including off Galicia56. Our results clearly confirm the existence of three spawning seasons, but spawning frequency could not be directly estimated and was therefore set as constant along the year and with female size . Obviously, this constrains our results on spawning duration by season and size class, given by potential/batch fecundity ratios and thereby not specifically attuned to the environmental influences that could certainly affect spawning rhythm.Our results clarify that hake egg productivity  changes with female size, but not substantially along the year. However, batch fecundity and length of spawning differed significantly by female size and, so far much less documented, also among seasons. These variations are crucial to our understanding of the reproductive strategy, including the realized egg production, of the stock. Temporal and regional variability in reproductive traits related to environmental conditions have been described for several fish species24, it would imply the presence of three spawning components within the same stock. The other option would be that each female takes part in each spawning season, i.e. they present three individual spawning periods, but this would lead to a considerably elevated energetic effort and the longest reproductive season reported for the genus Merluccius. Some of our results support this latter idea: i) PVO1-3 increase along the year as if they were to be used later, and ii) the amount of PVO4b-c in regressing and regenerating (RT) females is too high for them to be considered remnants. Although the level of (follicular cell) apoptosis or previtellogenic atresia were not assessed in this study, they are likely to be important processes34, at least for PVO1-3 where many of the oocytes “disappeared” to the next stage PVO4a, when contrasting immature and developing ovaries. The PVO4b-c oocytes could serve as surplus for the next spawning period occurring in a matter of weeks, which is supported by the observed dynamics of these PVO stages between RT and SC (spawning capable) females. More specifically, the RT-PVO4b level compares with the SC-PVO4c level, as does the RT-PVO4c level with the SC-VTO level. However, this way of thinking would be based on the idea that hake, living in a regime such as the Galician shelf ecosystem, should be able to spawn over a total of 7–8 months with an annual production of close to 20 million eggs for a 75-cm female. This possibility should not be entirely excluded, e.g. North Sea cod of the same size produced ≈4 million eggs in one single season, but this population of cod then waits a full year for the next season57. In any case, another clear option is a much shorter spawning duration for each female hake; it results in a more realistic individual fecundity and reproductive effort, but implies, as stated, the existence of different spawning components, i.e. winter-spring, summer and autumn spawners. Statutory ichthyoplankton surveys since 1999 show that species inhabiting the area in question are either winter or summer spawners, except for the mesopelagic fish, Maurolicus muelleri, which has a completely different life strategy, and the present object of study58. Also, the presence of three spawning seasons in southern European hake is reasonably well documented through this monitoring program. The existence of stable spawning components suggests some genetic differentiation, since a kind of reproductive isolation must occur. However, this is not a simple issue to address, as shown in genetic analyses conducted on, for example, spring and autumn spawning stocks of herring on both sides of the North Atlantic59, where genetic differences between components were certainly identified, but also associated with high gene flow. So, the alternative option of a single component, where each female spawns once a year for a short period, but at different times each year (which would result in three observed seasons) should be considered as biologically unrealistic. Nevertheless, the potential existence of several spawning components within the same stock, or even a single component with several spawning seasons, has a profound implication in fisheries assessment and management. Overfishing of one of the spawning components may occur if not managed properly. On the other hand, the stock-recruitment relationship is a cornerstone in fisheries assessment and estimation of recruitment is dependent on the spawning dynamics. Thus, further research must be performed to support sustainable exploitation.From a more speculative viewpoint, the findings in this article raise an important, but as yet unresolved, question about the actual duration of spawning or spawning period(s) of a single female within the same year. While this has been assumed to be 2–3 monthsde novo oocyte recruitment during spawning . However, the concept behind this thinking becomes blurred by our findings that the level of fecundity is set much earlier, meaning that hake shows a much more determinate, rather than an indeterminate, fecundity type at the “base line”, which suggests that the definition needs revising. Further to this, early oocyte recruitment patterns clearly differed throughout the year. Our work also points to a complex picture of environmental cues involved. Despite the many issues still unresolved, the fecundity pattern outlined here may well occur in a series of other teleosts. Thus, we question the well-established conceptions of primary and secondary growth oocytes being separate categories rather a continuum, and the concept of determinate versus indeterminate fecundity strategies limited to the spawning season. Hopefully, these results will stimulate new discussions within the scientific community to address teleost reproductive biology, particularly fecundity estimation, in a way that differs from that done today. This may have direct consequences in terms of fisheries advice and management with regard to estimation of SRP and SSB, and thereby potentially help improve sustainability.We found that, for European hake, oocyte recruitment – with direct consequences for the resulting egg production – occurs much earlier than previously thought, i.e. already during the gonadotropin-independent stage  and that the standing stock of PVO4b-VTO stages reflects reasonably well the potential fecundity of this species. If we adhere to the normal definition of a determinate and an indeterminate pattern, the European hake clearly falls into the latter category, as we found clear evidence of Merluccius merluccius) females (N = 2961) were collected monthly from commercial gillnet catches from the Galician shelf in the vicinity of the port of Laxe, Spain, from December 2011 to November 2012  was also registered. Assuming that the ovaries of European hake show an homogeneous oocyte distribution62, a cross section of the central part of each ovary was dehydrated, embedded in paraffin and histological sections of 4 µm were cut and stained with haematoxylin and eosin. The MAT was identified microscopically, based on the most advanced oocyte stage (MAO), on whether postovulatory follicles (POF) were present (spawning marker) or not, as well as on the type and amount of atresia, adapted from Hunter and Macewicz9 , specifically based on the location and shape of the circumnuclear ring (CNR), a cytoplasmic structure rich in organelles and RNA . Secondary growth (SG) stages were, following standard procedures, divided into cortical alveoli (CAO), and early (EVTO) and late vitellogenic oocytes (VTO), using zona radiata (chorion) appearance and the degree of accumulation and distribution of yolk granules as criteria, and, ultimately, migratory nucleus (MNO) and hydrated oocytes (HYO)24. Atresia was categorized into three types ; POFs were also registered24. Note: for clarity, we refer to stages for oocyte development, and phases for ovarian development, the latter being based on MAO, as described above.Histological sections were screened in order to classify the stages of oocyte development present . Oocyte size and shape were measured using a slightly modified version of the Elliptical oocytes project (https://sils.fnwi.uva.nl/bcb/objectj/examples/oocytes/), while grid counting was performed using the Weibel Grid Cell project (https://sils.fnwi.uva.nl/bcb/objectj/examples//Weibel/MD/weibel.html).For each ovary, ten micrographs of histological sections (fields) were taken at 10 × magnification, using a digital camera (DFC490 of 8.8 Mpx) mounted on a light microscope (Leica DM500B) with a resolution of 2.33 px/µm. To ensure no overlaps, these photos were taken from every second field starting from the ovarian wall and moving across the section, aided by the motorized Multistep module of the Leica Application Suite. Subsequent image analysis (see below) was carried out using the software ImageJi) (according to Delesse’s principle)64. The Vvi was computed on grid-overlaid histological sections as the ratio between grid points hitting stagei oocytes (or any of the other mentioned elements) and the total points hitting the sectioned tissue . A pilot study was performed in order to establish a compromise between accuracy and time consumption: (i) to avoid underestimation of Vvi of the earliest oocyte stages (PVOs), a first trial was performed on two females to find the most appropriate grid type, using two different set-ups, i.e. Grid A (240 points and 77.1 µm probe line length) and Grid B (370 points and 65.7 µm probe line length) to estimate Vvi, analyzing 10 fields per specimen in each case; (ii) a second trial was performed to define the number of counting fields per sample required to estimate Vvi reliably ), aiming at a deviation from normalized mean below ±0.05, studying ovaries from four females  using the selected grid type and ≤10 fields. These methodological issues have already been touched upon by other authors65, but given that, in this study, we focused on much smaller particles (down to ≈15 µm in oocyte diameter when embedded in paraffin), verification was needed to ensure a good level of precision and accuracy. Regarding trial (i), significant differences in PVO volume fraction appeared between Grid A and B (P < 0.01). Thus, we opted for the most conservative option, i.e. the denser grid (Grid B). Regarding the number of fields in trial (ii), the estimated deviation from the normalized mean for every oocyte stage in all specimens analysed stabilized at ±0.05 when seven fields were counted , missing oocytes and empty space; see Supplementary, Table ij) was calculated from the ratio between oocyte L and S axis, i.e. kij = Lij/Sij, and mean stageij-specific oocyte shape factor thereafter calculated for each female. Only oocytes sectioned through the nucleus were considered for measurements. The above-given number of stagei oocytes to be measured in order to estimate mean stagei-specific oocyte shape factor with a deviation from the normalized mean below ±0.05 was defined in a trial test on two females, where up to 15 stagei oocytes were measured /2. Stagei-specific mean volume-based oocyte diameter was then estimated as ODvi = [Σnij=1(ODij)3/ni)](1/3)16. Due to oocyte shrinkage during histological processing, a correction factor was applied to turn oocyte diameters into their initial, stabilized formaldehyde -fixed dimensions, as measured under laboratory conditions in whole mounts . We first tested the correction factor developed for resin-embedded ovarian tissue of European hake66. However, oocyte shrinkage varies with the embedding medium, being higher in paraffin than resin65. Since paraffin was used in this study, a second correction factor from a similar study on Thunnus alalunga using paraffin instead, was also taken into account65. Hence, we applied both correction factors in order to choose the one that gave the closest fit to the whole-mount recordings. Due to the high degree of shrinkage of hydrated oocytes (HYO) during histological processing and their resulting highly irregular shape, it was considered unfeasible to get reliable measurements of their oocyte size, thus ODi and OPDi were not estimated for this type of oocytes.For each specimen (j), short (S) and long (L) axes were measured on 10 oocytes of every oocyte stage (i) in histological slides. Individual oocyte shape factor  of oocytes present in the ovary by applying the refined formula of Korta et al.18:The number of oocytes per gram of ovary, known as the oocyte packing density (OPD)OPDij: Stagei-specific oocyte packing density by female (j)Vvij: Volume fraction of stagei oocytes by female (j)ρo: Specific gravity of the ovarykij: Mean shape factor of stagei oocytes by female (j)cODvii: Mean stagei volume-based oocyte diameter by female (j), corrected for shrinkage, cf.65.16, i.e. being set at 1.061 and 1.072 for ovaries showing PVO/MNO and CA/EVTO/VTO as the most advanced oocyte stage, respectively.The specific gravity of the ovary was obtained from Kurita and Kjesbui) in each ovary (j) was calculated from OPDij and the formalin-fixed gonad weight (GWfj) as:Shrinkage correction. To further clarify which shrinkage correction factor was the best one, we compared recent whole-mount estimates of batch fecundity (BF) for the same stock29 with our three estimations of the number of medium-late vitellogenic oocytes (VTO), the first being uncorrected for shrinkage (NOi) and the second and third being corrected for shrinkage using the two correction factors described above . The number of VTOs was assumed to reflect BF, as has been previously reported24. We found that mean cNOVTO values were closest to BF values at any total length of the female .Approaches for estimating NOi. As formalin-fixed ovary weight (GWf)  was estimated as NOi/TL3. To further validate this second approach, and concentrating on those specimens with EW values, the relative number of oocytes was also calculated as EW-based NOi = NOi/EW. The relationship between TL-based and EW-based NOi was extremely tight  = 2555, P < 0.001)  = 28.44, P < 0.001). Tests on the GW vs. TL relationships demonstrated unequal slopes among MAT  Eq.  both varGWf) Eq. , i.e. acTemporal trends. The temporal dynamics of oocyte production was analysed in three ways. First, an overall temporal analysis was conducted by estimating NOi values by month and oocyte stage, including other ovarian elements as well . These elements are known to be indicators of ovarian development. The noted variations were compared with trends in environmental variables , as well as with spawning fraction (SF) (see below). Second, a seasonal analysis was performed by estimating NOi values by ovarian phase (MAT) and oocyte stage within the respective spawning seasons identified. Information on duration and magnitude of spawning peaks was based on monthly SF, estimated as the proportion of the number of actively spawning females and the total number of sexually mature females by month, i.e. an average SF at the population level44. Note here that SF was estimated from the complete database of 2961 female specimens. This analysis resulted in the definition of three spawning seasons with different levels of spawning activity , and the spawning dynamics . To examine the influence of body size on oocyte recruitment dynamics, the analysis was performed using the absolute numbers of NOi  while considering four TL ranges . For each spawning season, the average NOi by oocyte stage and body size range was computed, pooling females in DV, SC and AS reproductive phases (Table EVTO and NOVTO (i.e. oocytes in various parts of vitellogenesis) showed comparable values in a given ovary, irrespective of the spawning season and female size. As we assumed NOVTO as a proxy for batch fecundity, this implied that the joint category of vitellogenic oocytes corresponded, in principle, to two, single batches and a mean batch fecundity was therefore estimated for each spawning season and female size range; BF = (NOEVTO + NOVTO)/2. We used this value to estimate the number of batches potentially existing in each oocyte stage and, from this, the average accumulated number of batches produced by a female in each season and size range. However, given that our sampling covered the whole spawning season, and given the strong spawning asynchrony among females , we assumed that the typical female analysed was in the middle of its spawning period . In consequence, each female had already released half of the batches.The number of oocytes in stage (www.indicedeafloramiento.ieo.es) using data on sea surface level pressure from the weather research and forecasting (WRF) operational model of Meteogalicia  for the region, between December 2011 and November 2012. Day length (number of daylight hours) at 43° 30′N 9°30′W was obtained from the Astronomical Applications Department of the U.S. Naval Observatory Data Service (http://aa.usno.navy.mil/index.php). The sea water temperature at the same geographical position at depth 50–350 m (monthly mean) was acquired from IBIRYS Regional High Resolution Reanalysis67 located at Copernicus Marine Environment Monitoring Services (http://www.copernicus.eu/) (product identifier: IBI_ REANALYSIS_PHYS_005_002). The above three selected abiotic factors were specifically used to explore impacts of environmental cues/drivers on European hake oocyte recruitment.Monthly upwelling indexes were provided by the Spanish Institute of Oceanography (https://www.r-project.org/) with P < 0.05 considered as a significant result. Differences in volume fraction (Vvi) between grid types were analyzed using paired t-test. The relationship between TL-based and EW-based NOi, was tested by a linear regression. ANCOVA was applied to contrast slope and intercept values for total length (TL) regressed on formalin-fixed gonad weight (GWf) split by ovarian phase (MAT). Relationships between potential fecundity and TL among spawning seasons were assessed by General Linear Models. All tests consulted were executed according to standard routines, as indicated in the various R scripts.All statistical analyses were performed using R version 3.2.3 (No specific permissions for sampling were required for this study as all the individuals sampled were obtained from commercial fishing following the local laws. Fish were purchased once the fishers processed them and analysed on board or right after landing. No protected species were sampled. No authorization or ethics board approval was required to conduct the study.Supplementary information


Macrophage activation syndrome (MAS) is a rare, potentially life-threatening, condition triggered by infections or flares in rheumatologic and neoplastic diseases. The mainstay of treatment includes high dose corticosteroids, intravenous immunoglobulins and immunosuppressive drugs although, more recently, a more targeted approach, based on the use of selective cytokines inhibitors, has been reported. We present the case of a two-year-old boy with 1-month history of high degree fever associated with limping gait, cervical lymphadenopathy and skin rash. Laboratory tests showed elevation of inflammatory markers and ferritin. By exclusion criteria, systemic onset Juvenile Idiopathic Arthritis (sJIA) was diagnosed and steroid therapy started. A couple of weeks later, fever relapsed and laboratory tests were consistent with MAS. He was promptly treated with high doses intravenous methylprednisolone pulses and oral cyclosporin A. One day later, he developed an acute myocarditis and a systemic capillary leak syndrome needing intensive care. Intravenous Immunoglobulin and subcutaneous IL-1-antagonists Anakinra were added. On day 4, after an episode of cardiac arrest, venous-arterial extracorporeal membrane oxygenation (VA-ECMO) was started. Considering the severe refractory clinical picture, we tried high dose intravenous Anakinra . This treatment brought immediate benefit: serial echocardiography showed progressive resolution of myocarditis, VA-ECMO was gradually decreased and definitively weaned off in 6 days and MAS laboratory markers improved. Our case underscores the importance of an early aggressive treatment in refractory life-threatening sJIA-related MAS and adds evidence on safety and efficacy of HDIV-ANA particularly in acute myocarditis needing VA-ECMO support. Macrophage activation syndrome (MAS) is a rare, potentially life-threatening complication of some rheumatologic diseases, such as systemic onset Juvenile Idiopathic Arthritis (sJIA), Kawasaki Disease (KD) and Systemic Lupus Erythematous (SLE), or a condition triggered by viral and or bacterial infections in predisposed individuals or associated with neoplastic disease . Early dWe describe the case of a child with sJIA complicated by severe MAS, Systemic Capillary Leak Syndrome (SCLS) and acute myocarditis, leading to distributive and cardiogenic shock, cardiac arrest needing cardiopulmonary resuscitation and veno-arterial Extracorporeal Membrane Oxygenation (VA-ECMO), who was successfully treated with high dose intravenous ANA (HDIV-ANA).A previously healthy two-year-old boy presented with 1 month history of fever associated with limping gait, cervical lymphadenopathy and evanescent skin rash. He was evaluated at a regional hospital where laboratory tests showed: WBC 25990/mm3 (N 18740/mm3); CRP 65 mg/L; ESR 68 mm/h; ferritin 1,259 ug/L; tryglicerides 1.5 mmol/L; AST 61 U/L, ALT 45 U/L. Echocardiography was normal. Bone marrow aspiration was negative for blasts. A short course of oral prednisone (1 mg/kg/day) was started with benefit on fever. However, upon steroid tapering, fever and limping reappeared. MRI showed synovial membrane hypertrophy and effusion in both hips. Therefore, according with ILAR criteria , sJIA waOn admission, physical examination revealed unremitting high-grade fever, erythematous skin rash on face and limbs and mild hepato-splenomegaly. No arthritis was detected.Laboratory tests showed: Hb 8.5 g/dl, PLT 44,000/mm3; FDP 1522 ug/L, fibrinogen 1.6 g/L, CRP 100 mg/L, AST 57 U/L, ALT 52 U/L; ferritin 2,200 ug/L; triglycerides 2.86 mmol/L. Suspecting an incipient MAS, high doses IV MDPN and oral Cyclosporin A  were started . 24 h laMAS is a life-threatening condition, most commonly reported as a complication of sJIA and triggered by infections in up to one-third of the patients . It is tIn our case, the intravenous ANA administration was a forced choice, partly due to the prominent generalized edema related to the SCLS and partly to the severe risk of bleeding, related both to MAS and ECMO. We now recognize that this choice has been successful. Along with the unexpected favorable result, no major adverse events were noticed, except for a transient neutropenia, already reported .Based on our experience, HDIV-ANA is a safe and effective treatment for refractory life-threatening sJIA-related MAS, even if complicated by acute fulminant myocarditis. Based on our positive experience, this therapeutic approach may be also considered in the current pandemic COVID-19 emergency where myocardial injury has been recently reported and where recent evidence showed interleukin-β-driven MAS-like complication, triggered by SARS-CoV-2 virus, as predictor of bad outcome –20.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.AM, GM, and FZ decided the personalized treatment, reviewed the literature about Anakinra use in MAS and received final approval by the hospital pharmacy, and general management department for its use. AM collected all data, conceptualized, and wrote the manuscript. FZ supervised the study team and critically reviewed the manuscript for important intellectual aspect of the work. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work. All authors took care of the patient during hospitalization, reviewed, and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Diffuse astrocytomas are the most aggressive and lethal glial tumors of the central nervous system (CNS). Their high cellular heterogeneity and the presence of specific barriers, i.e., blood–brain barrier (BBB) and tumor barrier, make these cancers poorly responsive to all kinds of currently available therapies. Standard therapeutic approaches developed to prevent astrocytoma progression, such as chemotherapy and radiotherapy, do not improve the average survival of patients. However, the recent identification of key genetic alterations and molecular signatures specific for astrocytomas has allowed the advent of novel targeted therapies, potentially more efficient and characterized by fewer side effects. Among others, peptides have emerged as promising therapeutic agents, due to their numerous advantages when compared to standard chemotherapeutics. They can be employed as (i) pharmacologically active agents, which promote the reduction of tumor growth; or (ii) carriers, either to facilitate the translocation of drugs through brain, tumor, and cellular barriers, or to target tumor-specific receptors. Since several pathways are normally altered in malignant gliomas, better outcomes may result from combining multi-target strategies rather than targeting a single effector. In the last years, several preclinical studies with different types of peptides moved in this direction, providing promising results in murine models of disease and opening new perspectives for peptide applications in the treatment of high-grade brain tumors. IDH1 and IDH2) gene mutations, this last group of tumors can be subclassified into IDH-mutant and IDH-wildtype categories, of which the first type is associated with better prognosis [Astrocytomas are the most common primary glial tumors of the central nervous system (CNS). In 2016, the World Health Organization (WHO) revised the 2007 CNS tumors classification, discerning astrocytomas into circumscribed (WHO grade I) and diffuse (WHO grades II-IV) subtypes based on their histology and molecular parameters. The first subtype includes benign astrocytic tumors, such as pilocytic astrocytomas, which are usually treatable with complete surgical resection. Conversely, the second group includes those gliomas that are more difficult to treat because of their heterogeneity and invasive growth. These include diffuse astrocytoma (grade II), anaplastic astrocytoma , and glioblastoma  [rognosis ,4.AA and GBM are two high-grade tumors. They are characterized by poor prognosis with a median survival of about 2–3 years and 12–15 months, respectively. Furthermore, they present with neurodegeneration, invasiveness, cytological pleomorphism, and increased mitotic activity. GBM exhibits also microvascular proliferation, necrosis, or both . PatientThe current treatment for AA and GBM consists of maximal surgical tumor resection, followed by chemotherapy with Temozolomide (TMZ) and focal radiotherapy. Furthermore, inpatient exercise rehabilitation programs after tumor resection were reported to have an important role to significantly improve neurocognitive and motor functions, which contribute to enhance the patients’ quality of life ,10. It iIn view of all these considerations, the development of novel therapeutic approaches, capable to overcome both the BBB and the tumor barrier, and able to inhibit tumor growth by selectively targeting GICs emerges as an urgent need. In the last few years, several studies have been performed to realize a genetic and lineage classification of GICs in order to design targeted and personalized therapies ,18,19. IIn this regard, the use of peptides for the treatment of a variety of diseases, including brain tumors, has been rapidly expanding. Some of these have already moved into Phase I/II clinical trials for the treatment of high-grade gliomas, showing promising results not only in terms of safety and tolerability, but also for their ability to reduce the tumor mass ,21,22.In this review, we provide an outline of the properties of different types of peptidic agents. Furthermore, we explore potential molecular targets for the treatment of high-grade astrocytomas using anti-tumor peptide therapeutics as well as peptide carriers able either to deliver anti-cancer molecules through cell and tissue barriers or to target tumor-specific receptors .Peptides are a novel class of compounds that can be used for the treatment of a wide range of pathological conditions, such as infections, diabetes, cardiovascular diseases, neurodegenerative disorders, and cancer. They are low molecular weight molecules, usually 10–50 amino acids long. The advantages of peptides are several-fold and include ease of synthesis, high specificity and activity, and low production cost. Furthermore, in the case of cancer, peptides result less immunogenic than recombinant monoclonal antibodies already used to target tumor cells with anti-cancer drugs . Since pIn vivo, peptides have poor stability due to their susceptibility to degradation by serum proteases. This short half-life prevents the development of drug resistance and increases their safety, though it may also reduce their efficacy. This constraint can be prevented by chemical modifications of the peptide sequence, for instance by integration of D-amino acids, cyclization, use of unnatural amino acids that are uncleavable by endogenous proteases, and blocking the access to the N- and C- terminal fragments . AnotherIn the case of brain tumor treatments, peptides can be employed in several ways, depending on their specific properties. According to their mechanism of action, they can be classified into two major groups. The first class includes peptides that are characterized by a direct mode of action. Some of these molecules have an intrinsic anti-cancer activity, so that they can be used as therapeutics, promoting the reduction of tumor growth by a variety of mechanisms. For instance, anti-microbial peptides (AMPs) are short cationic and hydrophobic molecules that play a cytotoxic action by targeting the negative charges of cancer cell membranes and inducing cell death through apoptosis or necrosis ,31. OtheIn the second group are peptide vaccines, which are characterized by an indirect mode of action. Cancer vaccines can be considered as active immunotherapies because they are based on the administration of tumor-associated antigens, which are specifically expressed in cancer tissues. The aim is to stimulate the immune system to react against the tumor . In thisAdvancements in the comprehension of molecular, cellular and genetic anomalies of various tumors have enabled the development of the “Atlas of Genetics and Cytogenetics in Oncology and Haematology”, a database listing several genes and proteins that are relevant for different types of cancer, including diffuse glioma. Some of these have been investigated as potential targets for small molecules or peptide vaccines, while others were specifically addressed with peptidic agents ,37,38,39Because most of the pathways that are altered in astrocytomas are primarily located within GICs, the main goal for the future is to develop strategies that specifically target this cellular subpopulation. Although several treatments conceived on this principle are still in the preclinical phase, there are solid promises for clinically relevant success.Here below, we report some detailed examples of glioma targets that can be specifically addressed by peptide-based drugs .In high-grade astrocytomas, such as GBM, an increased expression of the C-X-C chemokine receptor type 4 (CXCR4) has been reported. This can transduce growth signals in response to its protein ligand CXCL12. Clinically, CXCR4 expression levels in GBM correlate with increased tumor grade, aggressiveness and, consequentially, with a poor prognosis . A studyThese observations indicate that the CXCR4-CXCL12 pathway mediates survival and self-renewal in GICs with high selectivity, emerging as an attractive target for glioma-directed therapies. The CXCR4 antagonist that was most widely tested in clinical trials to reduce the growth of GBM, is the small molecule Plerixafor . Yet, itEpidermal growth factor (EGF) receptor (EGFR) is a transmembrane receptor tyrosine kinase involved in a wide variety of cellular processes and cancers, including GBM. EGFR is activated by a set of ligands, including EGF and transforming growth factor-alpha (TGF-α), which trigger its dimerization. The latter stimulates the autophosphorylation of the EGFR intracellular tyrosine kinase domain, leading to activation of numerous downstream signaling pathways, such as the phosphatidylinositol-3 kinase (PI3K)/AKT/rapamycin-sensitive mTOR-complex (mTOR) cascade. This signal transduction pathway is involved in various cellular functions, including cell cycle progression, differentiation, migration, and survival ,56. AKT It has been described that approximately half of the patients affected by GBM overexpresses EGFR, and 20–30% of them expresses the mutant truncated variant EGFRvIII. The latter is constitutively active and promotes persistent intracellular signaling, with consequent tumor growth, survival, invasion, and angiogenesis . Interestrans-activator of transcription (TAT) protein from HIV-1, since the large size and negative charges of siRNA hinder their cellular translocation. This system, named TAT-DRBD, was used to deliver EGFR and AKT siRNAs into an intracranial GBM mouse model to induce synthetic lethal RNA interference responses  channels . These cThese findings reveal that a combinatorial therapy, simultaneously targeting neo-angiogenesis and glioma cells, might represent an efficacious approach, since not only it would hinder oxygen and nutrients supply, but also kill tumor cells.Mounting evidence attributes GIC proliferation and resistance to deregulated pathways, such as Hedgehog (HH) signaling, which may be an effective target to improve GBM therapies. This signaling begins with the binding of HH ligands, more frequently sonic hedgehog (SHH), to the transmembrane receptor Patched, which initiates an intracellular signaling cascade that results in the activation of the family of Gli transcription factors. The upregulation of the HH/Gli1 pathway is associated with worse prognosis in GBM patients, because it is implicated in the regulation of cellular proliferation, survival, invasion, and angiogenesis ,67.2), isolated from phage display libraries -loaded nanoparticles (CK-NP-PTX) coated with a previously tested TTP, named CK peptide. This was composed of a VEGFR-2 targeting peptide (K237) bound, via a GYG linker, with a SHH targeting peptide , iThis drug delivery system was designed with the aim of increasing the therapeutic efficacy of glioma treatments, by simultaneously targeting the chemotherapeutic PTX to VM channels, tumor neovasculature, and GICs. To test the system, an intracranial glioma mouse model was injected intravenously with CK-NP-PTX. Researchers observed a selective accumulation of the compound around the vasculature as well as in the tumor parenchyma. This distribution determined a strong VM channels destruction, a significant apoptosis of glioma cells and an increase in medium survival time in treated mice when compared to controls .The MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway has been identified as a commonly dysregulated pathway in several cancers, including GBM. This cascade starts with the binding of a ligand to a transmembrane receptor tyrosine kinase and culminates with the phosphorylation through MEK of the final MAPK ERK. This latter translocates to the nucleus where it activates numerous transcription factors  involved in the regulation of a large variety of processes including cell proliferation, differentiation, migration, and apoptosis . Li and vβ3, are overexpressed in GBM, both on the surface of tumor cells as well as on angiogenic vessels. They contribute to angiogenesis and correlate with worse prognosis [vβ3 and αvβ5 integrins, preventing their interaction with ECM ligands. However, the promising results obtained in pre-clinical studies and in earlier clinical trials, were disconfirmed from the CENTRIC phase III trial that produced no survival benefit in treated patients when compared to controls [Integrins are a big family of cell adhesion transmembrane receptors composed of two associated α and β subunits, which directly bind several components of the ECM providing the adhesion required by tumor cells for their motility and invasion. Although integrins do not act as oncogenes, they cooperate with them or with receptor tyrosine kinases to increase tumorigenesis . Some tyrognosis ,72,73,74controls .vβ3 integrin antagonist RGD peptide. The co-delivery of PD0325901 and the RGD peptide allowed synergetic effects with inhibition of the ERK pathway, which is overactivated in GBM cells, and disruption of angiogenic signals in GBM tissue as well as inhibition of cell migration. Moreover, the RGD peptide permitted to deliver the MEK1/2 inhibitor to tumor cells and to the vasculature by integrin-targeted delivery, preventing off-target effects on healthy tissues . MDGI is a fatty acid-binding protein whose role in tumorigenesis is rather controversial and seems to vary in a cancer type-dependent manner. It can be linked to tumor-suppressor properties, e.g., in breast cancer , but alsTo this end, the group of Pirjo Laakkonen identified a novel synthetic tumor homing peptide, named CooP (ACGLSGLGVA), which specifically targets invasive tumor cells and the vasculature by binding to MDGI. They subsequently investigated the potential of CooP-targeted therapy to treat high-grade brain tumors. Thus, they developed a peptide-drug conjugate, named CooP-CPP-Cbl, in which the CooP peptide was covalently conjugated with the chemotherapeutic agent clorambucil (Cbl) and a CPP derived from the N-terminal part of the tumor suppressor protein p14ARF (MVRRFLVTLRIRRACGPPRVRV-NH2), to permit cellular internalization . To asseAs mentioned in the previous paragraphs, the most promising strategy to treat high-grade astrocytomas seems to be the simultaneous targeting of neovasculature/VM channels as well as glioma cells or GICs. Yet, an additional aspect that should be considered attentively is the poor penetration of most drugs in the tumor mass, due to the presence of a pathologic barrier constituted by ECM. The occurrence of this event is one of the causes of GBM recurrence . The ECMAmong ECM components, tenascin-C (TN-C) is a hexameric glycoprotein mainly expressed by neural and endothelial cells during embryogenesis. It is downregulated in adult healthy brains, but it becomes overexpressed in about 90% of GBMs. Tumor cells are the main source of TN-C release, and the intensity of its expression correlates with glioma grade and outcomes. TN-C is able to bind other ECM proteins as well as integrin receptors, thus influencing a number of cellular processes, such as cell migration, angiogenesis, and proliferation . For allNeuropilin-1 (NRP-1) is a multifunctional non-tyrosine kinase co-receptor expressed in many tissues, which binds a number of factors, including VEGF-A, Hedgehogs, TGFβ and EGF. It was shown to be highly expressed in GBM cells and neo-vasculature, where it regulates glioma growth, progression, and recurrence. The expression of NRP-1 correlates with glioma grade and poor patient prognosis ,81,82.In a recent study, Kang and collaborators developed a synergistic nanosystem constituted by PTX-containing nanoparticles coated with the synthetic Ft peptide (Ft-NP-PTX), which contains two sequences  and tLyp-1 (CGNKRTR) coupled via a cysteine) to simultaneously target TN-C and NRP-1, respectively. This system was designed to specifically circumvent the ECM barrier by targeting the glioma-related matrix component TN-C, and to concurrently achieve deep penetration in the glioma parenchyma, mediated by the over-expression of NRP-1 in glioma cells and vasculature . Mice beOver the last years, considerable progress has been made with regard to the development of pharmacological treatments for high-grade astrocytomas. Nonetheless, the prognosis of these types of cancer has not significantly improved and they remain the most aggressive and lethal tumors of the CNS. Even after total or subtotal surgical eradication of the tumor, followed by radiotherapy and concomitant adjuvant chemotherapy, the median survival does not exceed 3 years. There are many reasons for such a failure, including the critical brain localization and the lack of defined margins, which might hinder the total resection of the tumor mass. This event may contribute to its recurrence. Such disappointing results can be explained also with the structural complexity of malignant gliomas. In fact, it is well known that they are characterized by a great cellular heterogeneity and, within the tumor tissue, different sub-populations of poorly differentiated cells co-exist. Among these, GICs are the main responsible for tumor invasiveness and recurrence. These tumors are also supported by a complex network of blood vessels, including standard vasculature, which is constituted by endothelial cells, and VM channels, formed by glioma cells that are able to act as both endothelial and tumor cells. Finally, the complexity of high-grade gliomas can also be ascribed to the presence of an ECM-anomalous barrier that surrounds the tumor. This latter, together with the BBB, hampers the access of drug molecules to the tumor parenchyma.All these aspects make these types of cancer resistant to any kind of therapy. Furthermore, most chemotherapeutic agents are not selective for cancer cells, but they also damage healthy tissues, thus leading to diffuse adverse effects.In recent years, a significant number of key signaling molecules have been identified in many pathways, specifically altered in malignant gliomas, allowing the advent of more effective targeted and personalized therapies. In this framework, peptides have emerged as a novel class of etiology-based anti-cancer therapeutics that can be used as (i) pharmacologically active agents or (ii) carriers, either to facilitate the translocation of chemotherapeutics through brain, tumor, and cellular barriers, or to target tumor-specific molecular markers. This approach should make the therapy more specific, i.e., more effective and characterized by fewer side effects.Another aspect to consider carefully is that single-target approaches have not resulted in improved prognosis for patients affected by malignant gliomas. Better outcomes may result from combining multi-target strategies. For instance, peptide-based therapies can be designed to simultaneously target multiple effectors either on the same pathway or involved in different mechanisms and tumor compartments. To achieve this goal, it is necessary to target diverse markers on (i) ECM components, to enable the achievement of effective drug concentrations in the tumor mass; (ii) standard vasculature and VM channels, to remove nutrients and oxygen supply to the tumor; and (iii) glioma cells/GICs to kill those cells that become resistant to the hypoxic microenvironment and are responsible of metastasis formation. The successful elaboration of these approaches may enable, in the future, the development of effective personalized molecular therapies for high-grade astrocytomas.


Wolbachia strains are one of three endosymbionts associated with the insect vector of “Candidatus Liberibacter asiaticus,” Diaphorina citri Kuwayama (Hemiptera: Liviidae). We report three near-complete genome sequences of samples of Wolbachia from D. citri (wDi), with sizes of 1,518,595, 1,542,468, and 1,538,523 bp. Wolbachia strains are one of three endosymbionts associated with the insect vector of “Candidatus Liberibacter asiaticus,” Diaphorina citri Kuwayama (Hemiptera: Liviidae). We report three near-complete genome sequences of samples of Wolbachia from D. citri (wDi), with sizes of 1,518,595, 1,542,468, and 1,538,523 bp. Wolbachia is present in many insect species and can manipulate host reproduction via cytoplasmic incompatibility, male killing, and induction of parthenogenesis or feminization (Diaphorina citri Kuwayama (Hemiptera: Liviidae), the vector of the pathogen “Candidatus Liberibacter asiaticus,” associated with citrus greening disease, harbors three intracellular symbionts, including a strain of Wolbachia, wDi. Currently, the functional relationship of Wolbachia sp. strain wDi with D. citri and “Ca. Liberibacter asiaticus” is limited due to the unavailability of a genome assembly with no gaps. Here, we report three wDi genome sequences utilizing both long- and short-read sequencing methods. Wolbachia isolates were recovered from D. citri from an established laboratory culture collected in Polk County . Individual psyllids were placed on sterile diet rings for 2 days prior to Wolbachia extraction. The psyllids were surface sterilized and immersed in 1.0 ml of Schneider’s Drosophila (S2) medium . Next, individual psyllids were homogenized and centrifuged at 100 × g for 5 min. The supernatants were collected and centrifuged at 400 × g for 5 min. The pellets were resuspended with 1.0 ml of S2 medium. The samples were centrifuged at 100 × g for 5 min; then, supernatants were placed in new tubes and centrifuged at 4,000 × g for 5 min. The pellets were resuspended in 1.0 ml of S2 medium. After isolation, an individual wDi sample was inoculated into Drosophila S2 cells  and maintained in S2 medium containing 10% heat-inactivated fetal bovine serum , 50 units of penicillin, and 50 μg streptomycin sulfate  per ml (S2 complete medium) using the method described by Dobson et al.  to detect the generic variation of the bacteria in the host, using the protocol adopted from Rasgon et al. (The widespread endosymbiont nization . Diaphorhttps://github.com/lh3/seqtk) and Filtlong (https://github.com/rrwick/Filtlong). De novo assembly was performed separately using Canu v1.9 (The circular consensus sequences (CCS) generated from the PacBio raw reads were subjected to quality assessment and adaptor trimming using seqtk  (84.7%) (Drosophila melanogaster) (83.1%) (Wolbachia sp. strain wPip (from Culex pipiens) (86.3%) (https://phaster.ca/)  v4 using the bacteria_obd10 database  was util (84.7%) , wMel (f (83.1%) , and Wol (86.3%) . In addi (86.3%)  was done (86.3%)  (Table 1ter.ca/) . PHASTERCP051266, CP051265, and CP051264 corresponding to BioSample accession numbers SAMN14560310, SAMN14560311, and SAMN14560312, respectively, under the BioProject accession number PRJNA603775.The three genome sequences of wDi have been deposited in GenBank under the accession numbers