Post-Diapause DNA Replication during Oogenesis in a Capital-Breeding Copepod.

In high-latitude environments where seasonal changes include periods of harsh conditions, many arthropods enter diapause, a period of dormancy that is hormonally regulated. Diapause is characterized by very low metabolism, resistance to environmental stress, and developmental arrest. It allows an organism to optimize the timing of reproduction by synchronizing offspring growth and development with periods of high food availability. In species that enter dormancy as pre-adults or adults, termination of diapause is marked by the resumption of physiological processes, an increase in metabolic rates and once transitioned into adulthood for females, the initiation of oogenesis. In many cases, individuals start feeding again and newly acquired resources become available to fuel egg production. However, in the subarctic capital-breeding copepod Neocalanus flemingeri, feeding is decoupled from oogenesis. Thus, optimizing reproduction limited by fixed resources such that all eggs are of high quality and fully-provisioned, requires regulation of the number of oocytes. However, it is unknown if and how this copepod limits oocyte formation. In this study, the phase in oocyte production by post-diapause females that involved DNA replication in the ovary and oviducts was examined using incubation in 5-Ethynyl-2'-deoxyuridine (EdU). Both oogonia and oocytes incorporated EdU, with the number of EdU-labeled cells peaking at 72 hours following diapause termination. Cell labeling with EdU remained high for two weeks, decreasing thereafter with no labeling detected by four weeks post diapause, and three to four weeks before spawning of the first clutch of eggs. The results suggest that oogenesis is sequential in N. flemingeri with formation of new oocytes starting within 24 hours of diapause termination and limited to the first few weeks. Lipid consumption during diapause was minimal and relatively modest initially. This early phase in the reproductive program precedes mid-oogenesis and vitellogenesis 2, when oocytes increase in size and accumulate yolk and lipid reserves. By limiting DNA replication to the initial phase, the females effectively separate oocyte production from oocyte provisioning. A sequential oogenesis is unlike the income-breeder strategy of most copepods in which oocytes at all stages of maturation are found concurrently in the reproductive structures.

Em ambientes de alta latitude, onde as mudanças sazonais incluem períodos de condições adversas, muitos artrópodes entram em diapausa, um período de dormência regulado por hormônios. A diapausa é caracterizada por metabolismo muito baixo, resistência ao estresse ambiental e interrupção do desenvolvimento. Ele permite que um organismo otimize a reprodução sincronizando o crescimento e desenvolvimento da prole com períodos de alta disponibilidade de alimentos. Em espécies que entram em dormência como pré-adultos ou adultos, o término da diapausa é marcado pela retomada dos processos fisiológicos, um aumento nas taxas metabólicas e o início da oogênese. Em muitos casos, os indivíduos começam a se alimentar novamente e os recursos recém-adquiridos ficam disponíveis para abastecer a produção de ovos. No oceano subártico, a alimentação do copépode Neocalanus flemingeri é dissociada da oogênese e a reprodução é limitada por recursos fixos obtidos durante a pré-diapausa. No entanto, não se sabe como este copépode regula a formação de ovócitos para garantir que todos os óvulos sejam de alta qualidade e bem fornecidos. Neste estudo, a fase de produção de oócitos por fêmeas pós-diapausa foi examinada usando incubação em 5-Etinil-2'-desoxiuridina (EdU) para caraterizar a replicação do DNA no ovário e nos ovidutos. Tanto as oogônias quanto os oócitos incorporaram EdU, com o número de células marcadas atingindo o pico 72 horas após o término da diapausa. A marcação das células com EdU permaneceu alta por duas semanas, diminuindo na terceira e cessando na quarta semana. Desova da primeira ninhada de ovos ocorre três a quatro semanas depois. Os resultados sugerem que a oogênese é sequencial em N. flemingeri com a formação de novos oócitos começando dentro de 24 horas após o término da diapausa e limitado às primeiras semanas. O consumo de lipídios durante a diapausa foi mínima e relativamente modesto inicialmente. Essa fase inicial do programa reprodutivo precede a vitelogênese 2, quando os ovócitos aumentam de tamanho e acumulam reservas de vitelo e lipídios. Ao limitar a replicação do DNA à fase inicial, as fêmeas efetivamente separam a produção de oócitos do seu fornecimento. Uma oogênese sequencial é diferente da estratégia de reprodução da maioria dos copépodes que mantem oócitos em todos estágios de maturação nas estruturas reprodutivas.

Temperature compensation in the escape response of a marine copepod, Calanus finmarchicus (Crustacea).

Calanus finmarchicus, the dominant mesozooplankter of the North Atlantic, is an important food source for many fishes and other planktivores. This species, which has limited diel vertical migration, depends on its fast-start escape response to evade predators. It has myelinated neuronal axons, which contribute to its rapid and powerful escape response. The thermal environment that C. finmarchicus inhabits ranges from below 0 degrees C to 16 degrees C. Previous studies have shown that respiration, growth, and reproductive rates are strongly dependent on temperature, with Q10 > 2.5. A comparable dependence of the escape response could place the animal at higher risk for cold-compensated predators. Our work focused on the temperature dependence of the behavioral response to stimuli that mimic predatory attacks. We found that in contrast to other biological processes, all aspects of the escape response showed a low dependence on temperature, with Q10 values below 2. This low temperature dependence was consistent for escape parameters that involved neural as well as muscle components of the behavioral response. These findings are discussed in the contexts of the predator-prey relations of copepods and the thermal dependence of behavior in other taxa.

The need for speed. I. Fast reactions and myelinated axons in copepods.

A rapid and powerful escape response decreases predation risk in planktonic copepods. Calanoid copepods are sensitive to small and brief hydrodynamic disturbances: they respond with multiple nerve impulses to a vibrating sphere. Some species, such as Pleuromamma xiphias and Labidocera madurae, respond with very large spikes (1-4 mV), whereas maximum spike heights are an order of magnitude smaller in others, such as Undinula vulgaris and Neocalanus gracilis. A comparative study of the escape responses showed that all species reacted within 10 ms of the initiation of a hydrodynamic stimulus. However, U. vulgaris and N. gracilis had significantly shorter reaction times (minimum reaction times: 1.5 ms and 1.6 ms) than the other two, P. xiphias (6.6 ms) and L. madurae (3.1 ms). Examination of the first antenna and the central nervous system using transmission electron microscopy revealed extensive myelination of sensory and motor axons in the two species with the shorter reaction times. Axons of the other two species resembled typical crustacean unmyelinated fibers. A survey of 20 calanoids revealed that none of the species in two of the more ancient superfamilies possessed myelin, but myelination was present in the species from three more recently-evolved superfamilies.

The need for speed. II. Myelin in calanoid copepods.

Speed of nerve impulse conduction is greatly increased by myelin, a multi-layered membranous sheath surrounding axons. Myelinated axons are ubiquitous among the vertebrates, but relatively rare among invertebrates. Electron microscopy of calanoid copepods using rapid cryofixation techniques revealed the widespread presence of myelinated axons. Myelin sheaths of up to 60 layers were found around both sensory and motor axons of the first antenna and interneurons of the ventral nerve cord. Except at nodes, individual lamellae appeared to be continuous and circular, without seams, as opposed to the spiral structure of vertebrate and annelid myelin. The highly organized myelin was characterized by the complete exclusion of cytoplasm from the intracellular spaces of the cell generating it. In regions of compaction, extracytoplasmic space was also eliminated. Focal or fenestration nodes, rather than circumferential ones, were locally common. Myelin lamellae terminated in stepwise fashion at these nodes, appearing to fuse with the axolemma or adjacent myelin lamellae. As with vertebrate myelin, copepod sheaths are designed to minimize both resistive and capacitive current flow through the internodal membrane, greatly speeding nerve impulse conduction. Copepod myelin differs from that of any other group described, while sharing features of every group.

Mechanoreceptors in calanoid copepods: designed for high sensitivity.

The mechanoreceptors of the first antennae of Pleuromamma xiphias, a mesopelagic calanoid copepod, are critical for the detection of potential threats. These receptors exceed the physiological performance of other crustacean mechanoreceptors in sensitivity to water velocities as well as in frequency response. A study of these receptors was initiated to elucidate structure-function relationships. Morphologically, the receptors resemble the arthropod scolopidial organs by the presence of a scolopale tube. However, the rigidity of the copepod receptors greatly exceeds those described for crustaceans and other arthropods. The scolopale tube completely encloses the distal dendrites and it is firmly anchored to the cuticle. Microtubules are organized in register and arise from microtubule subfibers associated with crescent-shaped rods which extend from the basal body region to the setal socket. The distal dendrites are filled with a large number of cross-linked microtubules. Termination of the distal dendrites inside the lumen of the setae is gradual with a firm anchoring to the cuticle. A likely mechanism for mechanotransduction would involve a linkage between individual microtubules and mechano-gated channels in the dendritic membrane. The rigidity probably contributes to the high frequency sensitivity, and termination of the dendrite inside the seta increases the overall sensitivity of these receptors.

Rapid Jumps and Bioluminescence Elicited by Controlled Hydrodynamic Stimuli in a Mesopelagic Copepod, Pleuromamma xiphias.

Actively vertically migrating mesopelagic copepods are preyed upon by a wide variety of fishes and invertebrates. Their responses to predatory attacks include vigorous escape jumps and discharge of bioluminescent material. Escape jumps and bioluminescent discharges in the calanoid copepod Pleuromamma xiphias were elicited by quantified hydrodynamic disturbances. Brief weak stimuli (peak water velocity 64 +/- 21 {mu}m s-1) elicited weak (peak force 6.5 dynes) propulsive responses ("jumps") and no bioluminescence. Moderate stimuli (1580 +/- 780 {mu}m s-1) produced strong propulsive responses consisting of long trains of coordinated power strokes by the four pairs of swimming legs ("kicks"). Peak forces averaged 42 dynes. Strong stimuli (5520 +/- 3420 {mu}m s-1) were required to elicit both a jump and a bioluminescent discharge. In several cases, multiple stimuli were needed to evoke bioluminescence, given the limits on stimulus magnitude imposed by the apparatus. Repeated bioluminescent discharges could be evoked, but this responsiveness waned rapidly. Latencies for the jump response (14 +/- 4 ms) were shorter than for the accompanying bioluminescent discharge (49 +/- 26 ms). The higher threshold for eliciting bioluminescent discharge compared to escape jumps suggests that the copepods save this defense mechanism for what is perceived to be a stronger threat.

Dietary mustard, rape seed oils and selenium exert distinct effects on serum Se, lipids, peroxidation products and platelet aggregability.

OBJECTIVE: A degenerative cardiomyopathy (DCM) has been observed among mustard oil (MUST) users in China who eat 150 ml/week. Though the condition may be a selenium (Se) deficiency, MUST, rich in erucic acid (22:1), may bear a cardiotoxic or pro-oxidant substrate(s). The purpose of this feeding study was to compare the effects of feeding MUST, high erucic acid rape seed oil (HEAR), low erucic acid rape seed oil (LEAR) and corn oil, with or without Se addenda, on serum cholesterol, triglycerides, thiobarbituric acid reactive substances (TBARS), alpha-tocopherol, platelet aggregation and hepatic glutathione peroxidase (GSHPXase). Heart muscle pathology was evaluated. METHODS: Eighty male, weanling Wistar rats were divided into four dietary groups and fed AIN76A diet for 8 weeks with one of four oils: 1) MUST, 2) HEAR, 3) LEAR, or CORN oil. Half of each group received 0.43 mg/kg Se supplement. A purified diet was fed for 8 weeks. RESULTS: Neither Se nor oil type affected growth, though serum Se rose with dietary Se addendum (p < 0.01), and hepatic GSHPXase rose for each oil (p < 0.01). Se deprivation led to elevated serum cholesterol, except for the CORN oil group (p < 0.01). Only with LEAR did HDL cholesterol decrease. Serum triglycerides decreased with MUST and LEAR feedings (p < 0.05). In Se deficiency, HEAR led to elevated TBARS (p < 0.01), though not MUST, which contained twice as much alpha-tocopherol. Though supplementary Se protected the HEAR-fed animals from secondary peroxidation as TBARS (p < 0.01), the MUST group was not so protected. Ration Se also spared serum alpha-tocopherol for each diet group except the MUST/Se group (p < 0.05). MUST oil feeding resulted in increased platelet aggregation; MUST and HEAR resulted in increased platelet ATP release compared with CORN oil. CONCLUSION: Oxidative mechanisms may be involved in the DCM involving both Se limitation and pro-oxidant stress related to the usual intake of MUST. Human studies have been undertaken to test this thesis.

Effect of dietary menhaden, Canola and partially hydrogenated soy oil supplemented with vitamin E upon plasma lipids and platelet aggregation.

Male Fisher rats were fed chow diets for two weeks after which they were divided into seven groups of ten rats each and fed 20% Canola, 20% menhaden, 20% partially hydrogenated soy oil (PHSO) or chow only, with or without 500 mg/Kg dietary vitamin E in chow containing 2% cholesterol for six weeks. Triglycerides were lower in the menhaden group and were essentially the same in the E supplemented groups as in their unsupplemented cohorts. Plasma cholesterol was higher in the Canola, and lower in the menhaden, groups, compared to the PHSO group. Cholesterol was the same in the E supplemented groups as in their unsupplemented cohorts. Plasma thiobarbituric acid reactant substances (TBARS) were higher in the menhaden group, compared to the chow group. Vitamin E supplementation lowered TBARS in the menhaden and PHSO groups, compared to the unsupplemented cohorts. Collagen induced platelet aggregation was lower in both Canola and menhaden groups, compared to the PHSO group. Vitamin E supplementation lowered collagen induced platelet aggregation only in the PHSO group. Thrombin induced platelet aggregation was lower in the Canola group, compared to the PHSO group. Vitamin E supplementation did not affect thrombin induced platelet aggregation compared to unsupplemented cohorts. Plasma vitamin E levels were lowest in the menhaden supplemented group compared to all other groups not receiving E, suggesting a greater requirement for E in this group. Finally, vitamin E supplementation raised the plasma E levels in all groups except the menhaden group when compared to unsupplemented cohorts.

Standardization of incubation time and temperature for eight radioimmunoassays.

We wanted to know if incubation time and temperature for many radioimmunoassay methods could be standardized, to decrease assay time and so improve efficiency. Percentage binding was determined for triiodothyronine, thyroxine, digoxin, digitoxin, testosterone, aldosterone, estradiol, and diphenylhydantoin when the reaction mixtures were incubated at 6-8, 22, or 37 degrees C for various periods of time. In all methods tested, binding of antigen to antibody was greatest when the reaction mixtures were incubated at 6-8 degrees C, less at 22 degrees C, and least at 36 degrees C. Maximum binding occurred within 30-60 min at each temperature for all methods tested. Hence, it is possible to standardize the incubation time and temperature for these eight radioimmunoassay methods to 60 min at 6-8 degrees C.