Identification of late larval stage developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and steroid hormone signaling pathways.

Organisms in the wild develop with varying food availability. During periods of nutritional scarcity, development may slow or arrest until conditions improve. The ability to modulate developmental programs in response to poor nutritional conditions requires a means of sensing the changing nutritional environment and limiting tissue growth. The mechanisms by which organisms accomplish this adaptation are not well understood. We sought to study this question by examining the effects of nutrient deprivation on Caenorhabditis elegans development during the late larval stages, L3 and L4, a period of extensive tissue growth and morphogenesis. By removing animals from food at different times, we show here that specific checkpoints exist in the early L3 and early L4 stages that systemically arrest the development of diverse tissues and cellular processes. These checkpoints occur once in each larval stage after molting and prior to initiation of the subsequent molting cycle. DAF-2, the insulin/insulin-like growth factor receptor, regulates passage through the L3 and L4 checkpoints in response to nutrition. The FOXO transcription factor DAF-16, a major target of insulin-like signaling, functions cell-nonautonomously in the hypodermis (skin) to arrest developmental upon nutrient removal. The effects of DAF-16 on progression through the L3 and L4 stages are mediated by DAF-9, a cytochrome P450 ortholog involved in the production of C. elegans steroid hormones. Our results identify a novel mode of C. elegans growth in which development progresses from one checkpoint to the next. At each checkpoint, nutritional conditions determine whether animals remain arrested or continue development to the next checkpoint.

Feeding of anchovy Engraulis encrasicolus larvae in the northwestern Adriatic Sea in response to changing hydrobiological conditions

Results from depth integrated and vertically stratified plankton sampling in the northwestern Adriatic Sea were used for comparison of gut contents of larvae of European anchovy Engraulis encrasicolus with composition and concentration of potential prey in the plankton. Sampling was carried out over a grid of stations both before and after a period of increased wind mixing to investigate changes in food availability and larval feeding success. All larvae had empty guts soon after dusk, indicating daytime feeding and rapid gut clearance. With increasing larval length there was a greater percentage of specimens with empty guts, despite suitable food being available in the plankton for these larger larvae; this suggests differential gut evacuation during sampling-possibly related to the degree of gut development. Larval diet was principally the various developmental stages of copepods, especially calanoid and cyclopoid nauplii, which were preferentially selected by larvae, whereas selection was against harpacticoid nauplii. Lamellibranch larvae and Peridinium were generally abundant in the plankton, but were only present in the gut contents in any number when the preferred dietary organisms were present in the plankton at low concentrations. The number of food organisms in the gut contents increased with concentration of the preferred food organisms in the plankton up to a limit of similar to 50 organisms/l. Within the upper 18 m of the water column, there was a reduction in the proportion of larvae with food in their guts with increasing depth, irrespective of the vertical profile of food concentration. Following a period of wind mixing the composition of the plankton changed. This was reflected in the diet of anchovy larvae, which altered in parallel. There was also an overall 41% decrease in concentration of the preferred food particles of larvae in the plankton following the period of wind mixing, but larvae were still able to maintain their food intake. These results show that anchovy larvae can successfully adapt their diet to a changing prey field and suggest that in the conditions observed in the northern Adriatic, quite radical changes in the feeding environment were probably insufficient to affect overall larval mortality.

A synthesis of large-scale patterns in the planktonic prey of larval and juvenile cod (Gadus morhua)

Data from 40 published studies of the diet composition of larval and juvenile cod (Gadus morhua) from around the northern North Atlantic were summarized to assess generic patterns in ontogenetic and regional variability in the key prey. The results showed that larvae at the northern edge of the latitudinal range of cod depend primarily on development stages of the copepod Calanus finmarchicus, whilst those at the southern edge depend on Para- and Pseudocalanus species. Juvenile cod preyed on a wider range of taxa than larvae, but euphausiids were the main target prey. Analysis of regional variations in the relative abundances of C. finmarchicus and Para/Pseudocalanus spp. in the plankton, as estimated by the continuous plankton recorder (CPR) surveys, showed a similar geographical pattern to the larval cod stomach contents. Comparison of CPR data from the 1960s and 70s with data from the 1990s showed that the boundary between C. finmarchicus and Para/Pseudocalanus spp. dominance has shifted northwards on both sides of the Atlantic, whilst the abundance of euphausiids in the southern cod stock regions has declined. The results are discussed in relation to regional differences in the response of cod stocks to climate variability.

Embryo retrieval and kin recognition in an amphipod (Crustacea)

Active maternal care directed towards embryos within the brood pouch has been identified in amphipod crustaceans from harsh aquatic environments. This involves 'curl' and 'stretch' components and brood flushing that alters in distinct ways in response to developmental and environmental cues. However, a cost of active brood care in crustaceans is the susceptibility to embryo loss, this being further predisposed by the structure of the amphipod brood pouch. We found embryo retrieval by females of the rock-pool amphipod Apherusa jurinei, whereby females inserted experimentally offered embryos into their brood pouches. Females early in brood development retrieved embryos to a greater degree than both nonovigerous and later stage females. In this experiment, all offered embryos were from other females, indicating a motivation to retrieve embryos that often overrides any kin recognition. In a second experiment, we found kin discrimination, with both early stage and late stage females retrieving more of their own embryos than those from other females. Recognition was not simply of embryos of similar developmental stages. There were high levels of embryo cannibalism in both experiments, but females were significantly less likely to consume their own compared to foreign embryos. We thus further show that 'lower' crustaceans such as amphipods engage in elaborate active maternal care including kin recognition and discrimination. Their maternal behaviour appears to balance the costs and benefits of embryo retrieval, minimizing fitness reductions due to embryo loss and adoption of foreign embryos. (C) 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Embryonic and larval ecology and biochemistry of offshore decapods

Understanding the biology of offshore species is hardened by the difficulties of sampling in the deep-sea environment. Additionally, due to the vastness of the open ocean, knowledge of early life histories of pelagic larvae is still relatively scarce. In decapod species with bentho-pelagic lifestyle, the transition from life in the seafloor to the water column not only is associated with drastic morphological metamorphosis, but also with changes in behavior and feeding ecology. The purpose of the present thesis was to investigate physiological, biochemical and behavioral adaptation occurring during early development of such species. The Norway lobster, Nephrops norvegicus, and the crab Monodaeus couchi were used as a model as these two species are encountered off the NE Atlantic shelf at depth greater than 300 m. Chapter 1 introduces the challenges faced by both adult and larvae inhabiting such remote habitats, including the effect of food availability on development and oceanographic processes on dispersal and recruitment. The thesis follows early life histories, starting with within-brood variability in the fatty acid (FA) profile displayed by developing N. norvegicus embryos. There were no differences in the FA composition of embryos sampled from both sides of the brooding chamber in most females. However, all females exhibited significant differences in the FA profiles of embryos sampled from different pleopods. Potential causes for the variations recorded may be differential female investment during oocyte production or shifts in FA catabolism during the incubation period promoted by embryo’s location within the brooding chamber. Next, feeding rates and digestive enzymes activity of the early stage larvae was investigated in N. norvegicus. Both stages were able to maximize food intake when larvae were scarce and showed increased feeding rate following periods of starvation. Amylase activity indicated that carbohydrates are not the primary energy reserve and that feeding may be required soon after hatching to trigger amylase activity. Protease activity indicated that protein reserves are catabolized under starvation. These results indicate that larvae may maximize prey ingestion in the presence of plankton patches with higher food abundance and minimize the deleterious effects induced by previous periods of intermittent starvation or unsuitable prey densities/types. Additionally, changes in enzymatic activity may allow newly hatched N. norvegicus larvae to metabolize protein reserves to overcome short-term starvation. Vertical migration behavior and the influence of oceanographic properties were studied next. All zoeal stages of M. couchi displayed reverse diel vertical migration. Abundance of early stages was correlated with chlorophyll a levels. An ontogenic shift in vertical distribution explained the results; earlier zoeal stages remain in the food-rich upper water column while later stages migrate to the bottom for settlement. This vertical migration behavior is likely to affect horizontal distribution of larvae. Indeed, global current patterns will result in low inter-annual variations in decapod larvae recruitment, but short term variations such as upwelling events will cause deviation from the expected dispersal pattern. Throughout development, from the embryo to metamorphosis into benthic juvenile, offshore decapods face many challenges. For the developing individual survivorship will depend heavily on food availability but also on the reserves passed on by the mother. Even though vertical migration behavior can allow the larvae to take advantage of depth varying currents for transport, the effect of general circulation pattern will superimpose local current and influence feeding conditions and affect dispersal and recruitment.

Transient stimulation of glucose metabolism by insulin in the 1-day chick embryo.

Effects of insulin upon glucose metabolism were investigated in chick embryos explanted in vitro during the first 30 h of incubation. Insulin stimulated the glucose consumption of the chick gastrula (18 h) and neurula (24 h), but had no effect on the late blastula (0 h:laying) and on the stage of six to eight somites (30 h). The increase in glucose consumption concerned both the embryonic area pellucida (AP) and extraembryonic area opaca (AO). AP responded to a greater extent (50%) and at a lower range of concentrations (0.1-1.0 ng/ml) than AO (30%; 1-100 ng/ml). Insulin had no effect on the oxygen consumption of blastoderms, whereas it stimulated the aerobic lactate production (approximately 70% of the additional glucose consumption was converted to lactate). The nanomolar range of stimulating concentrations suggests that insulin has a specific effect in the chick embryo, and that it could modulate glucose metabolism in ovo as well. The transient sensitivity of the embryo to insulin is discussed in relation to behavior of mesodermal cells.

Pathogen-induced hatching and population-specific life-history response to water-borne cues in brown trout (Salmo trutta)

Hatching is an important niche shift, and embryos in a wide range of taxa can either accelerate or delay this life-history switch in order to avoid stage-specific risks. Such behavior can occur in response to stress itself and to chemical cues that allow anticipation of stress. We studied the genetic organization of this phenotypic plasticity and tested whether there are differences among populations and across environments in order to learn more about the evolutionary potential of stress-induced hatching. As a study species, we chose the brown trout (Salmo trutta; Salmonidae). Gametes were collected from five natural populations (within one river network) and used for full-factorial in vitro fertilizations. The resulting embryos were either directly infected with Pseudomonas fluorescens or were exposed to waterborne cues from P. fluorescens-infected conspecifics. We found that direct inoculation with P. fluorescens increased embryonic mortality and induced hatching in all host populations. Exposure to waterborne cues revealed population-specific responses. We found significant additive genetic variation for hatching time, and genetic variation in trait plasticity. In conclusion, hatching is induced in response to infection and can be affected by waterborne cues of infection, but populations and families differ in their reaction to the latter.

The effect of feeding regime on larval black fly (Diptera: simuliidae) primary head fan ray number

Identification of larval simuliids has always been difficult due to the morphological similarity many species bear to one another. For this reason all characters available have been drawn upon to aid in species identification, including head fan ray number. Even in light of an increasing body of anecdotal reports that head fan ray number is not fixed, it has continued to be used to aid species identification. In the current experiment simuliid larvae were reared under controlled laboratory conditions to last instar in one of three feeding regimes. Out of nine trials, the results of six showed a significant inverse relationship between feeding regime and head fan ray number. In addition to the laboratory experiments, larvae were also collected from the field over the course of the spring and summer, 1994. From these samples significant interspecific and intraspecific variations in head fan ray number were found both spatially and temporally within Algonquin Park. From these data it is concluded that head fan ray number for the species analysed is a developmentally plastic character, which varies in response to food availability. Furthermore, given the extreme variations in head fan ray number found in some species, I recommend that head fan ray number not be used as an aid to identification unless it can be shown to be a fixed character for the species in question.

Superovulation and embryo collection in nulliparous Boer goat does immunized against a recombinant ovine alpha-subunit inhibin

With the purpose of eliciting a superovulatory response, 12 adult nulliparous Boer goat does were actively immunized against a recombinant a-subunit of ovine inhibin (roIHN-alpha; two injections of 100 mg 4 weeks apart). Another 12 control Boer goat does were treated with physiological saline and acted as controls. One year later the immunized animals were boostered by the administration of another dose (100 mg) of the immunogen. Following treatment, blood samples were collected twice weekly for the periods of 16 and 12 weeks, respectively, to monitor the inhibin binding ability with the aid of a radio-tracer binding assay. Throughout the experiment, estrus detection was conducted twice daily with the aid of an aproned intact buck. From the first day after treatment to 48 h after standing estrus, ovarian activity was monitored daily by transrectal ultrasonography. On alternate estrous cycles, does were mated and 6 days later flushed transcervically to recover embryos. All goats treated with the roIHN-alpha produced antibodies reactive to the native bovine inhibin tracer-the titre increasing from 2.9 +/- 0.4 to a maximum of 21.9 +/- 2.9% binding after the second injection. The antibody titre gradually subsided over the next 16 weeks. The booster injection restored an elevated antibody titre (11.7 +/- 0.4%), which was maintained until the end of the sampling period 12 weeks later. In the control goats only trace amounts of antibody were recorded throughout the trial. In the roIHN-alpha-immunized goats the number of follicles reaching a diameter of > 4 mm was 14.6 +/- 1.2 per doe. A positive correlation was recorded between the follicle number and antibody titre (r=0.61; P < 0.01). The number of follicles ovulating per doe (6.9 +/- 0.7) followed the same tendency-however, the proportion decreased with increasing follicle numbers. A relatively weak correlation was recorded between the inhibin binding ability and number of ovulations (r=0.27; P < 0.05). In the control goats the majority (92%) of follicles exceeding 4 mm in diameter ovulated (2.5 +/- 0.1 follicles/doe). Embryo collection proved unsatisfactory (42% versus 39% recovery for immunized and control animals, respectively)-presumably because the uterine lumen of the nulliparous does was too narrow to permit effective flushing. In the group of immunized goats the occurrence of short estrous cycles (< 15 days) recorded was 34% versus only 6% in the controls. Overall, immunization of goats against roIHN-alpha led to an almost six-fold increase in number of ovarian follicles, a three-fold increase in ovulations and, despite the low recovery rate, a more than three-fold increase in ova or embryos recovered. It may be concluded that treatment of female goats with roIHN-alpha leads to an inhibin antibody response, accompanied by enhanced ovarian activity. The response was, however, accompanied by a large proportion of retained follicles and a high incidence of short estrous cycles. These problems need to be further investigated before rendering the method fit for application in embryo transfer programs in goats. (C) 2007 Elsevier B.V. All rights reserved.

Expression and regulation of Nkd-1, an intracellular component of Wnt signalling pathway in the chick embryo

The Wnt family of secreted signalling molecules control a wide range of developmental processes in all metazoans. The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement. Recent work has identified the protein Naked Cuticle to act as an intracellular switch to promote the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked Cuticle-1 (cNkd-1) and show that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly, we show that the expression of cNkd-1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

Synergistic effects of geographical strain, temperature and larval food on insecticide tolerance in Callosobruchus maculatus (F.)

This study investigated the multifactorial interaction of various environmental factors including 17 geographical strain (Brazil, Cameroon and Yemen strains), temperature, dose and larval food 18 (cowpea and mungbean) on the response of Callosobruchus maculatus adult to insecticide. All 19 the main factors, their two-way interactions and the four-way interaction had significant effects 20 on C. maculatus response to malathion (an organophosphate insecticide). However, the three-21 way interactions were not statistically significant (except strain x food x dose, P = 0.002). The 22 2 Brazil strain was the most responsive to temperature irrespective of the larval food type. The 23 impact of food type differs from one strain to the other, for instance, the food that imparts higher 24 tolerance in a strain might reduce the tolerance in another. Likewise, the hierarchy of tolerance 25 among the cowpea-reared strains (Brazil > Cameroon > Yemen) was totally different from the 26 mungbean-reared strains (Cameroon > Yemen > Brazil). The reasons for these differences were 27 discussed in the light of their impact on C. maculatus management. The management of both C. 28 maculatus and development of resistance could be complex, hence, the states of a variety of 29 environmental factors need to be considered. This is necessary in order to maximize management 30 success of this bruchid especially in tropical/subtropical developing countries.

Human-animal interaction, stress, and embryo production in Bos indicus embryo donors under tropical conditions

This study investigated the effect of human-animal interaction (HAI) and the stress response on the quality of embryo production in superovulated Nelore (Bos indicus) cattle, under tropical conditions. Thirty-two females underwent a superovulation protocol for 5 days. Cortisol concentrations were determined in blood plasma collected on days 0, 4, and 5. Artificial insemination was performed on days 4 and 5, and nonsurgical embryo flushing on day 11. Embryo production and viability were determined. Human stimulation, animal behaviors, accidents, and handling time were recorded to assess HAI. Cattle age was negatively correlated with accidents, frequency of aversive behaviors, and negative stimuli by stockperson during transit through corral compartments to receive superovulation treatments. The factor analysis revealed two distinct groups. The first group was called stressed and had higher cortisol concentration than the nonstressed group, 16.0 +/- 2.1 and 12.5 +/- 1.0 ng/mL, respectively. Comparisons between these groups showed that the frequency of voice emissions by the stockperson and the number of accidents were higher in the stressed group, and also, the mean handling time was longer in the stressed group than for the nonstressed. As a result, viability rate of the embryos was 19% lower in the stressed group (P < 0.05). This indicates that intensive negative HAI is likely related to stress, which affects embryo production in a superovulation program.

Recruitment of the infaunal bivalve Soletellina Alba (Lamark,1818) (Bivalvia: Psammobiidae) in response to different sediment types and water depths within the intermittently open Hopkins River estuary

Studies examining recruitment processes for soft-sediment macroinvertebrate fauna in intermittent estuaries are rare and most studies of active habitat selection have been tested in the laboratory rather than the field. The present field study examined whether recruitment of the infaunal bivalve Soletellina alba was influenced by water depth and sediment particle size in the intermittent Hopkins River estuary, southern Australia. The number of recruits in sediment trays differed between water depths, but active habitat selection was not evident across treatments of varying sediment particle size. The use of sediments with varying particle sizes also provided an opportunity to identify potential discontinuities in body-size distributions of recruits associated with varying habitat architecture. The length (mm) of recruits was converted to the same scale used to express sediment particle size (i.e. phi units: phi = − log2 of sediment particle size). The size of recruits differed across water depths, but did not differ across treatments with fine (phi = 3) versus coarse (phi = 1) sediment, and no relationships were apparent between bivalve size and sediments consisting of varying particle size. These patterns of recruitment do not correspond with the distribution of adult S. alba within the Hopkins River estuary. Previous sampling has shown that abundances of juvenile and adult S. alba are variable across time, site and water depth, but are often greater at the deeper water depth (1.05 m below the Australian Height Datum). However, recruitment during the present study was greatest at the shallower water depth (0.05 m below AHD), and the apparent absence of active habitat selection suggests that the distribution of adults is unlikely to be attributable to differences in recruitment associated with sediments of varying particle size.

Nutritional related aspects of the artificial propagation and larval rearing of Macquarie perch, Macquaria Australasica (Cuvier, 1830)

Studies the underlying role of nutrition in the lack of response of captive fish to hypophysation. Aspects studied include morphological characteristics, histology of ovaries, proximate analysis, fatty and amino acid profiles of oocytes, muscle, liver and diets of wild and tank-reared fish, egg and larval quality, amino acid composition of eggs and larvae at different developmental stages, larval feeding and hormone treatments.

Heat tolerance, behavioural temperature selection and temperature-dependent respiration in larval Octopus huttoni

This study reports temperature effects on paralarvae from a benthic octopus species, Octopus huttoni, found throughout New Zealand and temperate Australia. We quantified the thermal tolerance, thermal preference and temperature-dependent respiration rates in 1-5 days old paralarvae. Thermal stress (1°C increase h^-1) and thermal selection (~10-24°C vertical gradient) experiments were conducted with paralarvae reared for 4 days at 16°C. In addition, measurement of oxygen consumption at 10, 15, 20 and 25°C was made for paralarvae aged 1, 4 and 5 days using microrespirometry. Onset of spasms, rigour (CT_max) and mortality (upper lethal limit) occurred for 50% of experimental animals at, respectively, 26.0±0.2°C, 27.8±0.2°C and 31.4±0.1°C. The upper, 23.1±0.2°C, and lower, 15.0±1.7°C, temperatures actively avoided by paralarvae correspond with the temperature range over which normal behaviours were observed in the thermal stress experiments. Over the temperature range of 10°C-25°C, respiration rates, standardized for an individual larva, increased with age, from 54.0 to 165.2nmol larvae^-1h^-1 in one-day old larvae to 40.1-99.4nmol h^-1 at five days. Older larvae showed a lesser response to increased temperature: the effect of increasing temperature from 20 to 25°C (Q₁₀) on 5 days old larvae (Q₁₀=1.35) was lower when compared with the 1 day old larvae (Q₁₀=1.68). The lower Q₁₀ in older larvae may reflect age-related changes in metabolic processes or a greater scope of older larvae to respond to thermal stress such as by reducing activity. Collectively, our data indicate that temperatures >25°C may be a critical temperature. Further studies on the population-level variation in thermal tolerance in this species are warranted to predict how continued increases in ocean temperature will limit O. huttoni at early larval stages across the range of this species.