Viability of Embryo Sacs and Fruit Set in Different Plum (Prunus domestica L.) Cultivars Grown under Norwegian Climatic Conditions

Compatibility and synchrony between specialized tissues of the pistil, female gametophytes and male gametophytes, are necessary for successful pollination, fertilization, and fruit set in angiosperms. The aim of the present work was to study the development and viability of embryo sacs, as well as fertilization success, in relation to the fruit set of the cultivars ‘Mallard’, ‘Edda’, ‘Jubileum’, and ‘Reeves’, under specific Norwegian climatic conditions. Emasculated, unpollinated, and open-pollinated flowers were collected at the beginning of flowering, and on the 3rd, 6th, 9th, and 12th days after flowering, from all four plum cultivars over two years (2018/2019). Ovaries were dehydrated, embedded in paraffin wax, sectioned, stained, and observed under a light microscope. Results showed the existence of synchronization between successive phases in the development of the embryo sac and individual phases of flowering. All plum cultivars had higher percentages of viable embryo sacs, fertilized embryo sacs, and fruit set in 2018 than in 2019. These differences may be related to the very low temperatures during the post-full-flowering period in 2019, and to the low adaptation of some studied cultivars to unfavorable conditions. In our study, the cultivar ‘Jubileum’ showed the highest percentage of viable embryo sacs, fertilized embryo sacs, and fruit set compared to other cultivars, i.e., the best low-temperature adaptation.

Sperm Numbers as a Paternity Guard in a Wild Bird

Sperm competition is thought to impose strong selection on males to produce competitive ejaculates to outcompete rival males under competitive mating conditions. Our understanding of how different sperm traits influence fertilization success, however, remains limited, especially in wild populations. Recent literature highlights the importance of incorporating multiple ejaculate traits and pre-copulatory sexually selected traits in analyses aimed at understanding how selection acts on sperm traits. However, variation in a male’s ability to gain fertilization success may also depend upon a range of social and ecological factors that determine the opportunity for mating events both within and outside of the social pair-bond. Here, we test for an effect of sperm quantity and sperm size on male reproductive success in the red-back fairy-wren (Malurus melanocephalus) while simultaneously accounting for pre-copulatory sexual selection and potential socio-ecological correlates of male mating success. We found that sperm number (i.e., cloacal protuberance volume), but not sperm morphology, was associated with reproductive success in male red-backed fairy-wrens. Most notably, males with large numbers of sperm available for copulation achieved greater within-pair paternity success. Our results suggest that males use large sperm numbers as a defensive strategy to guard within-pair paternity success in a system where there is a high risk of sperm competition and female control of copulation. Finally, our work highlights the importance of accounting for socio-ecological factors that may influence male mating opportunities when examining the role of sperm traits in determining male reproductive success.

Infestation by Octolasmis species (Lepadiformes, Poecilasmatidae) of the marine crab Lupocycloporus gracilimanus (Decapoda, Portunidae) from India

The portunid crab Lupocycloporus gracilimanus from Digha, Bay of Bengal, India was investigated for an infestation by the lepadomorph cirriped Octolasmis spp. About 93% of the epibionts examined were found attached to the surface of the carapace, abdomen and legs, and these were identified as Octolasmis warwicki (EOW). Only 7% of the epibionts were attached to the branchial lamellae, and these were identified as Octolasmis angulata (EOA). Twenty-seven O. warwicki were also found to have conspecific dwarf males (CDM) attached to the scutum: probably an adaptive strategy to maximize the total insemination. Both the epibionts and the CDM showed spatial preference for the posterior mesobranchial region of the carapace. Most of the CDM (27%) were seen as a single attachment on the epibiont. The distance between the epibiont and its nearest neighbour (DNN) was found to be strongly and negatively correlated (, ) with the number of epibionts, indicating its preference to stay in an intermediate-sized group rather than remain solitary, which probably would have facilitated the evolution of dwarf males (CDMs). A higher percentage of epibionts were observed to have attached CDMs when the DNN increased, and above the 25 mm DNN, all epibionts were having CDM attachments, which is believed to be a strategy to maximize the fertilization success of CDMs by reducing sperm competition. A strong, positive correlation (, ) was observed between the carapace width of the host crab and the numbers of the infesting epibionts, proving that the larger crabs are more susceptible to the infestation compared to the smaller ones. The sex ratio of the host crab was 1 : 5.5 (Male : Female), nevertheless, only the females were infested by the epibionts. The percentage of the prevalence, relative abundance and mean intensity of EOW infestation were recorded as 46.15%, 182.05% and 394.44%, respectively. None of the crabs had serious external or internal infestations that seemed to hinder the activities of the animal. This is the first report of the infestation of these Octolasmis species on the portunid crab Lupocycloporus gracilimanus from India.

Delta Smelt (Hypomesus transpacificus) Exhibit Wide Variation in Spawning Behavior: An Investigation of Substrate Type, Diel Timing, and Participants

Studies of cultured populations can be invaluable to the conservation of imperiled species for which little is known and whose very low abundance in the wild can preclude studies with robust sample sizes. For example, in endangered delta smelt (Hypomesus transpacificus), conservation measures developed to mitigate population decline are hindered by a lack of knowledge regarding spawning behavior. However, the availability of a cultured population allows important questions about spawning to be addressed experimentally. We examined the effect of substrate type on spawning behavior and egg fertilization success in cultured H. transpacificus under laboratory conditions. We also examined the diel frequency of spawning and the sex and number of spawning participants. We found that the frequency of spawns and resulting egg fertilization success did not differ between sand, gravel, and control (acrylic) substrates. We also demonstrated that spawning was predominantly nocturnal, though rare diurnal spawns were observed. Of the five mature males and five ripe females available within a given trial, spawns included up to seven participants, but most frequently occurred between a single female and one or two males. Our results highlight the wide behavioral variation exhibited by H. transpacificus during spawning, particularly in terms of substrate use, diel timing, and participants. These findings provide the most detailed and experimentally robust data regarding H. transpacificus spawning behavior to date and thereby provide ongoing conservation efforts with much-needed information.

Genetic coordination of sperm morphology and seminal fluid proteins promotes male reproductive success in Drosophila melanogaster

Spermatozoal morphology is highly variable both among and within species and in ways that can significantly impact fertilization success. In Drosophila melanogaster, paternity success depends on sperm length of both competing males and length of the female's primary sperm storage organ. We found that genes upregulated in long sperm testes are enriched for lncRNAs and seminal fluid proteins (Sfps). Transferred in seminal fluid to the female during mating, Sfps are secreted by the male accessory glands (AG) and affect female remating rate, physiology, and behavior with concomitant advantages for male reproductive success. Despite being upregulated in long sperm testes, they have no known function in testis tissue. We found that Sex Peptide and ovulin (Acp26Aa) knockouts resulted in shorter sperm, suggesting that Sfps may regulate sperm length during spermatogenesis. However, knockout of AG function did not affect sperm length, suggesting that AG expression has no influence on spermatogenic processes. We also found that long sperm males are better able to delay female remating, suggesting higher Sfp expression in AG. These results might suggest that long sperm males have a double advantage in sperm competition by both delaying female remating, likely through transfer of more Sfps, and by resisting sperm displacement. However, we also found that this extra advantage does not necessarily translate to more progeny or higher paternity success. Thus, we found that multiple components of the ejaculate coordinate to promote male reproductive success at different stages of reproduction, but the realized fitness advantages in sperm competition are uncertain.

Seed Paternity Analysis Using SSR Markers to Assess Successful Pollen Donors in Mixed Olive Orchards

The olive tree (Olea europaea L.) is a wind-pollinated crop that exhibits an extreme alternate bearing habit. To improve fruit set, several methods have been used to determine the most successful compatible combinations of cultivars. In this study, priority is given to seed paternity analysis based on simple sequence repeats (SSRs), microsatellite markers used for the identification of potential pollen donors of cultivar ‘Oblica’ in a mixed olive orchard during two consecutive years. Seven microsatellite primers were successfully used to examine the paternity of olive embryos from ‘Oblica’ mother trees. Embryos were considered as a product of self-fertilization if only maternal alleles were present, but not a single case of self-fertilization was found among all the embryos analyzed. Two dominant pollen donors were not the closest nor the cultivars with the highest number of trees in the orchard, suggesting that cross-compatibility may have a key role in determining pollen donor success. In our earlier studies, pollen tube growth and fertilization success correlated with fruit set when controlled crosses between cultivars were performed; however, some discrepancy might appear compared to paternity analyses when mother trees have a free choice among different pollen sources from cultivars growing in their surroundings.

Genetic Variation in Released Gametes Produces Genetic Diversity in the Offspring of the Broadcast Spawning Coral Acropora Tenuis

All coral species in the genus Acropora are broadcast-spawning hermaphrodites. Fertilization in the ocean requires sufficient numbers of gametes from conspecifics and the contact time for fertilization is thought to be limited by the rapid diffusion of sperm. Many studies have reported a positive correlation between sperm concentration and fertilization success, but it is not clear how released gametes are fertilized in nature (in situ) and how genetic diversity arises at a fertilization event. To elucidate this, we analyzed the changes in sperm concentration in situ after spawning and genotyped sperm and fertilized eggs from seawater using seven microsatellite (MS) markers. This showed that the sperm concentration in situ was suboptimal (<106 sperm/mL), but most of the eggs were fertilized. MS genotyping showed that the alleles of released sperm were diverse and those alleles also appeared in the fertilized eggs. In addition, the MS fragment peak height in released sperm, which presumable reflects the allele frequency of the sperm, was positively correlated with the allele frequencies of the fertilized eggs. Collectively, the genetic diversity of colonies with high fecundity dictates the genetic diversity of their descendants.

High-speed camera recordings uncover previously unidentified elements of zebrafish mating behaviors integral to successful fertilization

The mating behavior of teleost fish consists of a sequence of stereotyped actions. By observing mating of zebrafish under high-speed video, we analyzed and characterized a behavioral cascade leading to successful fertilization. When paired, a male zebrafish engages the female by oscillating his body in high frequency (quivering). In response, the female pauses swimming and bends her body (freezing). Subsequently, the male contorts his trunk to enfold the female’s trunk. This behavior is known as wrap around. Here, we found that wrap around behavior consists of two previously unidentified components. After both sexes contort their trunks, the male adjusts until his trunk compresses the female’s dorsal fin (hooking). After hooking, the male trunk slides away from the female’s dorsal fin, simultaneously sliding his pectoral fin across the female’s gravid belly, stimulating egg release (squeezing/spawning). Orchestrated coordination of spawning presumably increases fertilization success. Surgical removal of the female dorsal fin inhibited hooking and the transition to squeezing. In a neuromuscular mutant where males lack quivering, female freezing and subsequent courtship behaviors were absent. We thus identified traits of zebrafish mating behavior and clarified their roles in successful mating.