Long-term survey of sea turtles (Caretta caretta) reveals correlations between parasite infection, feeding ecology, reproductive success and population dynamics.

Long-term monitoring of host-parasite interactions is important for understanding the consequences of infection on host fitness and population dynamics. In an eight-year survey of the loggerhead sea turtle (Caretta caretta) population nesting in Cabo Verde, we determined the spatiotemporal variation of Ozobranchus margoi, a sanguivorous leech best known as a vector for sea turtle fibropapilloma virus. We quantified O. margoi association with turtles' δ15N and δ13C stable isotopes to identify where infection occurs. We then measured the influence of infection on reproduction and offspring fitness. We found that parasite prevalence has increased from 10% of the population in 2010, to 33% in 2017. Stable isotope analysis of host skin samples suggests transmission occurs within the host's feeding grounds. Interestingly, we found a significant interaction between individual size and infection on the reproductive success of turtles. Specifically, small, infected females produced fewer offspring of poorer condition, while in contrast, large, infected turtles produced greater clutch sizes and larger offspring. We interpret this interaction as evidence, upon infection, for a size-dependent shift in reproductive strategy from bet hedging to terminal investment, altering population dynamics. This link between infection and reproduction underscores the importance of using long-term monitoring to quantify the impact of disease dynamics over time.

Distribution of genetic diversity reveals colonization patterns and philopatry of the loggerhead sea turtles across geographic scales.

Understanding the processes that underlie the current distribution of genetic diversity in endangered species is a goal of modern conservation biology. Specifically, the role of colonization and dispersal events throughout a species' evolutionary history often remains elusive. The loggerhead sea turtle (Caretta caretta) faces multiple conservation challenges due to its migratory nature and philopatric behaviour. Here, using 4207 mtDNA sequences, we analysed the colonisation patterns and distribution of genetic diversity within a major ocean basin (the Atlantic), a regional rookery (Cabo Verde Archipelago) and a local island (Island of Boa Vista, Cabo Verde). Data analysis using hypothesis-driven population genetic models suggests the colonization of the Atlantic has occurred in two distinct waves, each corresponding to a major mtDNA lineage. We propose the oldest lineage entered the basin via the isthmus of Panama and sequentially established aggregations in Brazil, Cabo Verde and in the area of USA and Mexico. The second lineage entered the Atlantic via the Cape of Good Hope, establishing colonies in the Mediterranean Sea, and from then on, re-colonized the already existing rookeries of the Atlantic. At the Cabo Verde level, we reveal an asymmetric gene flow maintaining links across island-specific nesting groups, despite significant genetic structure. This structure stems from female philopatric behaviours, which could further be detected by weak but significant differentiation amongst beaches separated by only a few kilometres on the island of Boa Vista. Exploring biogeographic processes at diverse geographic scales improves our understanding of the complex evolutionary history of highly migratory philopatric species. Unveiling the past facilitates the design of conservation programmes targeting the right management scale to maintain a species' evolutionary potential.

Circadian pacemaker coupling by multi-peptidergic neurons in the cockroach Leucophaea maderae.

Lesion and transplantation studies in the cockroach, Leucophaea maderae, have located its bilaterally symmetric circadian pacemakers necessary for driving circadian locomotor activity rhythms to the accessory medulla of the optic lobes. The accessory medulla comprises a network of peptidergic neurons, including pigment-dispersing factor (PDF)-expressing presumptive circadian pacemaker cells. At least three of the PDF-expressing neurons directly connect the two accessory medullae, apparently as a circadian coupling pathway. Here, the PDF-expressing circadian coupling pathways were examined for peptide colocalization by tracer experiments and double-label immunohistochemistry with antisera against PDF, FMRFamide, and Asn(13)-orcokinin. A fourth group of contralaterally projecting medulla neurons was identified, additional to the three known groups. Group one of the contralaterally projecting medulla neurons contained up to four PDF-expressing cells. Of these, three medium-sized PDF-immunoreactive neurons coexpressed FMRFamide and Asn(13)-orcokinin immunoreactivity. However, the contralaterally projecting largest PDF neuron showed no further peptide colocalization, as was also the case for the other large PDF-expressing medulla cells, allowing the easy identification of this cell group. Although two-thirds of all PDF-expressing medulla neurons coexpressed FMRFamide and orcokinin immunoreactivity in their somata, colocalization of PDF and FMRFamide immunoreactivity was observed in only a few termination sites. Colocalization of PDF and orcokinin immunoreactivity was never observed in any of the terminals or optic commissures. We suggest that circadian pacemaker cells employ axonal peptide sorting to phase-control physiological processes at specific times of the day.

The circadian biology of the marbled crayfish.

The parthenogenetic marbled crayfish (Procambarus spec.) has recently been introduced as a new preparation for neuroethological studies. Since isogeneity apparently limits inter-individual variation, this otherwise typical decapod species may be especially valuable for circadian studies. Locomotor activity of isolated marbled crayfish and agonistic activity of small social groups maintain circadian rhythmicity in constant darkness. As potential signals of circadian time information, levels of 5HT, N-acetylserotonin and melatonin were determined in brains of marbled crayfish at different daytimes. However, location and structural organization of crustacean circadian pacemakers are still elusive. Immunocytochemical and backfill studies in the marbled crayfish revealed neural structures that may correspond to portions of circadian pacemaker systems in the insect optic lobe. Position and additional chemical contents in two pigment-dispersing hormone-expressing neuron groups resembled insect pigment-dispersing factor-expressing cells in the lamina and the accessory medulla, a neuropil discussed as center for integration of timing information. Here, we discuss new findings about the possible organization of the circadian system of the marbled crayfish in the light of current knowledge about circadian clocks in crustacea.

Circadian regulation of agonistic behavior in groups of parthenogenetic marbled crayfish, Procambarus sp.

Crustaceans have frequently been used to study the neuroethology of both agonistic behavior and circadian rhythms, but whether their highly stereotyped and quantifiable agonistic activity is controlled by circadian pacemakers has, so far, not been investigated. Isolated marbled crayfish (Procambarus spec.) displayed rhythmic locomotor activity under 12-h light:12-h darkness (LD12:12) and rhythmicity persisted after switching to constant darkness (DD) for 8 days, suggesting the presence of endogenous circadian pacemakers. Isogenetic females of parthenogenetic marbled crayfish displayed all behavioral elements known from agonistic interactions of previously studied decapod species including the formation of hierarchies. Groups of marbled crafish displayed high frequencies of agonistic encounters during the 1st hour of their cohabitation, but with the formation of hierarchies agonistic activities were subsequently reduced to low levels. Group agonistic activity was entrained to periods of exactly 24 h under LD12:12, and peaks of agonistic activity coincided with light-to-dark and dark-to-light transitions. After switching to DD, enhanced agonistic activity was dispersed over periods of 8-to 10-h duration that were centered around the times corresponding with light-to-dark transitions during the preceding 3 days in LD12:12. During 4 days under DD agonistic activity remained rhythmic with an average circadian period of 24.83 +/- 1.22 h in all crayfish groups tested. Only the most dominant crayfish that participated in more than half of all agonistic encounters within the group revealed clear endogenous rhythmicity in their agonistic behavior, whereas subordinate individuals, depending on their social rank, initiated only between 19.4% and 0.03% of all encounters in constant darkness and displayed no statistically significant rhythmicity. The results indicate that both locomotion and agonistic social interactions are rhythmic behaviors of marbled crayfish that are controlled by light-entrained endogenous pacemakers.

Pigment-dispersing hormone (PDH)-immunoreactive neurons form a direct coupling pathway between the bilaterally symmetric circadian pacemakers of the cockroach Leucophaea maderae.

Circadian locomotor activity rhythms of the cockroach Leucophaea maderae are driven by two bilaterally paired and mutually coupled pacemakers that reside in the optic lobes of the brain. Transplantation studies have shown that this circadian pacemaker is located in the accessory medulla (AMe), a small neuropil of the medulla of the optic lobe. The AMe is densely innervated by about 12 anterior pigment-dispersing-hormone-immunoreactive (PDH-ir) medulla (PDHMe) neurons. PDH-ir neurons are circadian pacemaker candidates in the fruitfly and cockroach. A subpopulation of these neurons also appears to connect both optic lobes and may constitute at least one of the circadian coupling pathways. To determine whether PDHMe neurons directly connect both accessory medullae, we injected rhodamine-labeled dextran as neuronal tracer into one AMe and performed PDH immunocytochemistry. Double-labeled fibers in the anterior, shell, and internodular neuropil of the AMe contralaterally to the injection site showed that PDH-ir fibers directly connect both accessory medullae. This connection is formed by three anterior PDHMe neurons of each optic lobe, which, thus, fulfill morphological criteria for a direct circadian coupling pathway. Our double-label studies also showed that all except one of the midbrain projection areas of anterior PDHMe neurons were innervated ipsilaterally and contralaterally. Thus, anterior PDHMe neurons seem to play multiple roles in generating circadian rhythms. They also deliver timing information output and perform mutual pacemaker coupling in L. maderae.

Ultrastructure of pigment-dispersing hormone-immunoreactive neurons in a three-dimensional model of the accessory medulla of the cockroach Leucophaea maderae.

Locomotor activity rhythms of the cockroach Leucophaea maderae are orchestrated by two bilaterally symmetric, mutually coupled, circadian pacemakers. They lie in the optic lobes of the brain and are confined to the accessory medulla (AMe), ventro-medially to the medulla. The AMe is innervated by approximately 12 pigment-dispersing hormone (PDH)-immunoreactive anterior medulla neurons (PDHMe), which are circadian pacemaker candidates in the fruitfly and the cockroach. We have developed a three-dimensional computer model of the AMe and associated structures as a framework for neuroanatomical studies. Our greatly improved understanding of this structure in space has allowed us further to subdivide the anterior PDHMe into three subgroups, i.e., large, medium-sized, and small anterior PDHMe. The synaptic connections of two of these subgroups have been examined within subcompartments of the AMe by light and electron microscopy. The large, intensely staining, anterior PDHMe contain medium-sized dense-core vesicles and form input and output synapses with profiles densely filled with clear vesicles primarily in the anterior and shell neuropil of the AMe. The medium-sized anterior PDHMe contain large dense-core vesicles and constitute input and output synapses either with profiles being densely filled with clear vesicles, or with profiles containing granular dense-core vesicles. The small, weakly staining anterior PDHMe belong to a morphological group different from the large and medium-sized PDHMe and cannot be further identified at the electron-microscopic level because of their weak PDH immunoreactivity.

Neural organization of the circadian system of the cockroach Leucophaea maderae.

The cockroach Leucophaea maderae was the first animal in which lesion experiments localized an endogenous circadian clock to a particular brain area, the optic lobe. The neural organization of the circadian system, however, including entrainment pathways, coupling elements of the bilaterally distributed internal clock, and output pathways controlling circadian locomotor rhythms are only recently beginning to be elucidated. As in flies and other insect species, pigment-dispersing hormone (PDH)-immunoreactive neurons of the accessory medulla of the cockroach are crucial elements of the circadian system. Lesions and transplantation experiments showed that the endogeneous circadian clock of the brain resides in neurons associated with the accessory medulla. The accessory medulla is organized into a nodular core receiving photic input, and into internodular and peripheral neuropil involved in efferent output and coupling input. Photic entrainment of the clock through compound eye photoreceptors appears to occur via parallel, indirect pathways through the medulla. Light-like phase shifts in circadian locomotor activity after injections of gamma-aminobutyric acid (GABA)- or Mas-allatotropin into the vicinity of the accessory medulla suggest that both substances are involved in photic entrainment Extraocular, cryptochrome-based photoreceptors appear to be present in the optic lobe, but their role in photic entrainment has not been examined. Pigment-dispersing hormone-immunoreactive neurons provide efferent output from the accessory medulla to several brain areas and to the peripheral visual system. Pigment-dispersing hormone-immunoreactive neurons, and additional heterolateral neurons are, furthermore, involved in bilateral coupling of the two pacemakers. The neuronal organization, as well as the prominent involvement of GABA and neuropeptides, shows striking similarities to the organization of the suprachiasmatic nucleus, the circadian clock of the mammalian brain.

Ectopic transplantation of the accessory medulla restores circadian locomotor rhythms in arrhythmic cockroaches (Leucophaea maderae).

The presence of an endogenous circadian clock in the brain of an animal was first demonstrated in the cockroach Leucophaea maderae. However, the clock's cellular basis remained elusive until pigment-dispersing hormone-immunoreactive neurons, which express the clock genes period and timeless in Drosophila, were proposed as pacemaker candidates. In several insect species, pigment-dispersing hormone-immunoreactive neurons are closely associated with the accessory medulla, a small neuropil in the optic lobe, which was suggested to be a circadian clock neuropil. Here, we demonstrate that ectopic transplantation of adult accessory medulla into optic lobe-less cockroaches restores circadian locomotor activity rhythms in L. maderae. All histologically examined cockroaches that regained circadian activity regenerated pigment-dispersing hormone-immunoreactive fibres from the grafts to original targets in the protocerebrum. The data show that the accessory medulla is the circadian pacemaker controlling locomotor activity rhythms in the cockroach. Whether pigment-dispersing hormone-immunoreactive neurons are the only circadian pacemaker cells controlling locomotor activity rhythms remains to be examined.

Optic lobe commissures in a three-dimensional brain model of the cockroach Leucophaea maderae: a search for the circadian coupling pathways.

The circadian rhythm of locomotor activity in the cockroach Leucophaea maderae is controlled by bilaterally symmetric, apparently directly coupled, circadian pacemakers in the optic lobes. Strong evidence predicts that ventromedial to the medulla, the accessory medulla with associated pigment-dispersing hormone-immunoreactive neurons is this circadian clock. In search for direct coupling pathways between both clocks, we performed horseradish peroxidase backfills from one optic stalk as well as dextran and horseradish peroxidase injections into one accessory medulla. Seven commissures with projections in the contralateral optic lobe were identified and reconstructed. Three of these commissures connected both accessory medullae. Two of these resembled the arborization pattern of the pigment-dispersing hormone-immunoreactive neurons, which are circadian pacemaker candidates in insects. This finding suggests that some of these pacemaker candidates form a direct circadian coupling pathway. For better visualization of reconstructed commissures, we implemented the reconstructions into a three-dimensional model of the cockroach brain.