Progress of developmental programme during the last larval instar of Bombyx mori: Relationships with food intake,ecdysteroids and juvenile hormone

The progress of developmental programme in the epidermal cells of last instar larvae of Bombyx mori was determined by ecdysteroid injections in normal and in JH-treated larvae. To clarify the importance of food intake in the control of development, starved animals were also used.The instar begins with a period during which the larval programme is expressed: this occurs in the presence of 20-hydroxyecdysone. Epidermal cells can thereafter secrete pupal cuticle after ecdysteroid injection although the larval programme is normally still present. During the last period only pupal characters can be expressed either in normal or in 20-hydroxyecdysone-injected larvae.These different developmental phases are not correlated with obligatory and facultative feeding periods.Transition from the first to the second phases is correlated with the absence of JH effects on pupal genes. JH applications during the second period, however, prevent the expression of pupal characters after 20-hydroxyecdysone injection. Thus, during this period, the pupal programme is not stabilized. Cellular reprogramming itself occurs at the onset of the last developmental period and is probably under the control of ecdysteroids.     

Larval size relative to larval feeding, cannibalism of larvae, egg or adult female size and larval–adult setal patterns among 13 phytoseiid mite species

Phytoseiid mite larvae vary in size and feeding type. We compared larval size to feeding by larvae, cannibalism of larvae by adult females, egg and adult female size and the setae lengths of larvae and adults among 13 species. There was no relationship between size of larvae and either feeding by larvae or cannibalism of larvae by adult female mites. Correlations were highest between larval size as measured by idiosoma plus extended leg lengths and adult female size of idiosoma plus extended leg lengths (r²=0.746), while next highest was larval idiosoma length and adult female idiosoma length (r²=0.662) and then larval idiosoma length and egg length (r²=0.579). Based on idiosoma length, Phytoseiulus persimilis had the largest larvae (non-feeding) among species and Euseius finlandicus had the smallest larvae (obligatory feeding). However, based on idiosoma length plus extended leg length, obligatory feeding larvae (on pollen or mites) of E. finlandicus and Euseius hibisci were largest and facultative feeding larvae (on mites) of Neoseiulus californicus and obligatory feeding larvae (on mites) of Galendromus occidentalis were the smallest. Among species with non- or facultative feeding larvae, Amblyseius andersoni and Neoseiulus barkeri had larger larvae and Typhlodromus pyri and Neoseiulus fallacis had smaller larvae when leg lengths were included in larval size. Setae lengths of larvae versus adult females (after adjustment for body sizes) showed high correlation for j6 (r²=0.942) and s4 (r²=0.854), but low correlation for larval Z4 versus adult female Z4 (r²=0.084) or Z5 (r²=0.063). Overall, larval morphological traits were most closely correlated to traits of other life stages, although for setae there were some exceptions. Differences in the functions of setae j6, s4 and Z4 in the larva versus adult female are discussed.     

Ontogenetic patterns of cold-hardiness and glycerol production in Sarcophaga crassipalpis

Developmental patterns of low-temperature tolerance and glycerol production were determined for larval, pupal and adult stages of the flesh fly Sarcophaga crassipalpis Macquart (Diptera: Sarcophagidae). Both diapause and non-diapause-destined flies were reared at relatively high temperatures, 20° or 25°C, prior to testing. Cold tolerance was greatest for diapause pupae aged 12–35 days after pupariation. Among non-diapause-destined flies, pupae exhibited a greater level of low temperature tolerance than larvae or adults. Although diapause pupae were more tolerant than non-diapause pupae maximal cold tolerance was not attained in either group until 10 days after pupariation. Non-diapause-destined feeding and wandering larvae had higher glycerol levels than larvae destined for diapause. During the first 6 weeks after pupariation glycerol titres increased steadily in diapause pupae. Rapid loss of glycerol is associated with the termination of pupal diapause.     

Effects of prehatching salinity and initial larval biomass on survival and duration of development in the zoea 1 of the estuarine crab, Chasmagnathus granulata, under nutritional stress

The effects of individual larval biomass, and salinity experienced during embryogenesis (i.e., prehatching salinity) on starvation tolerance and growth of zoea 1 of the estuarine crab (Chasmagnathus granulata) were evaluated in laboratory experiments. Freshly hatched zoeae 1 were obtained from broods maintained at three salinities (15‰, 20‰ and 32‰), and cultured at 20‰ under different initial feeding periods and subsequent food deprivation (“point of reserve saturation” experiment: PRS) or under initial periods of food deprivation and subsequent feeding (point of no return experiment: PNR). Another group of larvae were used for determination of biomass (dry weight, carbon, and nitrogen) of zoea 1.Larval survival and duration of development depended on the length of feeding period: no larvae reached the second instar under complete starvation; survival was higher and duration of development shorter as the feeding period lengthened. After different initial feeding periods (PRS experiment), zoeae 1 that hatched from eggs incubated at the prehatching salinities of 15‰ and 20‰ showed higher survival and shorter duration of development than those at 32‰. Prehatching salinity also affected the amount of reserves accumulated during the first 2 days after hatching, with larvae from 15‰ and 20‰ showing the highest percentage of total accumulation of carbon and nitrogen. Initial larval biomass did not affect survival, but it had a slight effect on duration of development, with larger larvae (in terms of biomass) developing faster. After different initial starvation periods (PNR experiment), prehatching salinity did not affect survival, but it affected duration of development: larvae from 15‰ and 20‰ reached the second instar earlier. Variability in survival and duration of development was explained in part by among-brood variability in initial larval biomass: larvae with higher biomass showed higher survival and shorter duration of development. Thus, C. granulata, survival and duration of development under food stress depend on the interaction between environmental conditions experienced before and after hatching (pre- and posthatching factors, respectively).     

Influence of starvation on larval development of Carcinus maenas L. (Decapoda : Portunidae)

Lethal and sublethal effects of particular starvation events were investigated in larvae of Carcinusmaenas L. Mean survival times of continuously starved zoeae-1 were approximately twice the normal stage duration (12, 18, 25°C), and both increased with falling temperatures. At 6°C zoea-1 was unable to develop to stage-2. No larva retained the ability for successful further development if starved for half the stage duration time and was then refed. The zoea-1 larvae had to feed for at least 20 % of the normal stage duration for some larvae to moult to zoea-2. Some initial feeding was necessary to start zoea-1 development. Beyond a certain point of energy and accumulation of reserves development of the larvae seems to continue regardless of feeding rates. The demands for larval feeding correspond very well with the larval moulting cycle. Larvae of C. maenas proved to be well adapted to natural shortage of food.     

The impact of food type, temperature and starvation on larval development of Balanus amphitrite Darwin (Cirripedia: Thoracica)

The impact of diatom food species (Chaetoceros calcitrans and Skeletonema costatum), temperature and starvation on the larval development of Balanus amphitrite was evaluated. Starvation threshold levels for different ages of larvae (0- to 5-day-old) fed with C. calcitrans and S. costatum and then starved at 5, 15 and 25 °C temperature were estimated as ultimate recovery hour (URH; denoting the starvation point in hours at the end of which larvae can recover and continue development). Effect of temperature on starvation threshold varied significantly with larval age and food species. The URH declined with larval age at 5 °C, but not at 15 and 25 °C. The URH and grazing rates were high for early instars fed on C. calcitrans, and for advanced instars fed on S. costatum. Carbon gain through feeding was maximum for 2-day-old larvae when fed with C. calcitrans and decreased with larval age. However, when fed with S. costatum carbon gain increased with larval age. This confirms that with development the utility of food types changes. The differences in the carbon gain can be attributed to differences in grazing rate due to variations in the size of the diatom cells, larval intersetular distance, diatom sinking rate and the photo-taxic behavior of larvae. Molting was observed at times when larvae were undergoing starvation and this could be viewed as stress-induced molting, and it differed with the larval age and food organisms.     

The relationship between egg size and the duration of the facultative feeding period in marine invertebrate larvae

Feeding larvae of marine invertebrates fuel development from both endogenous egg energy and exogenous energy obtained from the planktonic environment. Although both sources of energy likely influence certain larval stages, only the effects of exogenous food have been well studied. Despite the lack of research on the effects of egg size on larval stages, investigators have hypothesized that egg size influences the duration of the facultative feeding stage—the stage in which larvae can feed but do not have to because development is still being fueled by egg energy. To test this hypothesis, we investigated six species of sand dollars with different sized eggs and quantified the duration of the larval facultative feeding period of each species by comparing when fed and starved larvae diverged in size. Regardless of whether phylogeny was taken into account, the duration of the facultative feeding period was positively correlated with egg size. We further determined that our conclusions were not sensitive to either our estimation of the duration of the facultative feeding period, or the branch lengths of the phylogeny we used. This relationship is likely a result of larger eggs being provisioned with more energy, and may affect how well larvae can cope with natural variability in food concentrations. Furthermore, our results support an assumption of a theoretical model developed to understand the evolution of different life-history strategies in marine invertebrate larvae, which suggests that this relationship has important evolutionary consequences.     

Effects of egg size reduction and larval feeding on juvenile quality for a species with facultative-feeding development

In free-spawning marine invertebrates, larval development typically proceeds by one of two modes: planktotrophy (obligate larval feeding) from small eggs or lecithotrophy (obligate non-feeding) from relatively large eggs. In a rare third developmental mode, facultative planktotrophy, larvae can feed, but do not require particulate food to complete metamorphosis. Facultative planktotrophy is thought to be an intermediate condition that results from an evolutionary increase in energy content in the small eggs of a planktotrophic ancestor. We tested whether an experimental reduction in egg size is sufficient to restore obligate planktotrophy from facultative planktotrophy and whether the two sources of larval nutrition (feeding and energy in the egg) differentially influence larval survival and juvenile quality. We predicted, based on its large egg size, that a reduction in egg size in the echinoid echinoderm Clypeaster rosaceus would affect juvenile size but not time to metamorphosis. We reduced the effective size of whole (W) zygotes by separating blastomeres at the two- or four-cell stages to create half- (H) or quarter-size (Q) “zygotes” and reared larvae to metamorphosis, both with and without particulate food. Larvae metamorphosed at approximately the same time regardless of food or egg size treatment. In contrast, juveniles that developed from W zygotes were significantly larger, had higher organic content and had longer and more numerous spines than juveniles from H or Q zygotes. Larvae from W, H and Q zygotes were able to reach metamorphosis without feeding, suggesting that the evolution of facultative planktotrophy in C. rosaceus was accompanied by more than a simple increase in egg size. In addition, our results suggest that resources lost by halving egg size have a larger effect on larval survival and juvenile quality than those lost by withholding particulate food.     

Activities of natural methyl farnesoids on pupariation and metamorphosis of Drosophila melanogaster

Methyl farnesoate (MF) and juvenile hormone (JH III), which bind with high affinity to the receptors USP and MET, respectively, and bisepoxy JH III (bisJH III) were assessed for several activities during Drosophila larval development, and during prepupal development to eclosed adults. Dietary MF and JH III were similarly active, and more active than bisJH III, in lengthening larval development prior to pupariation. However, the order of activity was changed (JH III > bisJH III > MF) with respect to preventing prepupae from eclosing as normal adults, whether administered in the larval diet or as topically applied at the white puparium stage. If endogenous production of all three larval methyl farnesoids was suppressed by a strongly driven RNAi against HMGCR in the corpora allata cells, most larvae did not attain pupariation. Farnesol (which has no demonstrated life-necessary function in larval life except in corpora allata cells as a precursor to methyl farnesoid biosynthesis) when incorporated into the diet rescued attainment of pupariation in a dose-dependent manner, presumably by rescuing endogenous production of all three hormones. A more mild suppression of endogenous methyl farnesoid production enabled larval attainment of pupariation. However, in this background dietary MF had increased activity in preventing puparia from attaining normal adult eclosion. The physiological relevance of using exogenous methyl farnesoids to block prepupal development to normally eclosed adults was tested by, instead, protecting in prepupae the endogenous titer of methyl farnesoids. JH esterase normally increases during the mid-late prepupal stage, presumably to clear endogenous methyl farnesoids. When JH esterase was inhibited with an RNAi, it prevented attainment of adult eclosion. Cultured adult corpora allata from male and female Aedes aegypti released both MF and JH III, and the A. aegypti nuclear receptor USP bound MF with nanomolar affinity. These A. aegypti data support the use of Drosophila as a model for mosquitoes of the binding of secreted MF to USP.     

The contribution of the facultative feeding period to echinoid larval development and size at metamorphosis: a comparative approach

Planktotrophic invertebrate larvae have two dissociable stages during development, a facultative feeding period, whose length is determined by the amount of maternal provisioning in the egg, and an obligate feeding period, whose length is determined by the quantity of exogenous energy needed to reach metamorphic competence. Here we set out to experimentally test the impact of feeding during the facultative feeding period at two food concentrations (limiting and nonlimiting) on larval development time and juvenile quality. We used two closely related subtropical sand dollar species that differ in the quantity of maternal investment for these comparisons: Leodia sexiesperforata (large egg, long facultative feeding period) and Mellita tenuis (small egg, short facultative feeding period). We found that feeding during the facultative period accelerates development to metamorphosis only in M. tenuis and only at the high food ration. Feeding during the facultative feeding period had no effect on development time for M. tenuis at a food limiting concentration and for L. sexiesperforata at either food concentration. Furthermore, we found feeding during the facultative period to significantly increase quantity of carbohydrates and lipids at metamorphosis only for M. tenuis in nonlimiting food concentration. Thus, our data reveal a two-fold benefit of the facultative feeding period for a poorly provisioned species under high food conditions but little effect on a well-provisioned species. We discuss our results in reference to McEdward's [McEdward, L.R., 1997. Reproductive strategies of marine benthic invertebrates revisited: facultative feeding by planktotrophic larvae. Am. Nat. 150, 48-72] facultative feeding model.     

Effects of food availability on larval development in the slipper limpet Crepidula onyx (Sowerby)

Effects of food availability on the larval survival and development of Crepidula onyx were studied in four experiments by feeding the larvae with different concentrations of the chrysophyte Isochrysis galbana and by starving the larvae for different periods of time. Food concentration had a clear impact on the survival, growth and development time of C. onyx veligers. Larval development occurred only at 10⁴ cells ml⁻¹ and higher algal concentrations. No shell increment was detected in the veligers cultured for 12 days at 10² cells ml⁻¹I. galbana or the blank control. At 10³ cells ml⁻¹, there was only a slight increase in shell length over 12 days. At 10⁴ cells ml⁻¹, about 40% of the larvae became competent in 18 days. At 10⁵ and 10⁶ cells ml⁻¹, more than 90% of the larvae reached competence in 7 days. Initial starvation negatively affected the larval development, but the sensitivity differed among parameters measured on day 5: lower survivorship was detected only for larvae that had suffered 3 days or longer initial starvation, whereas one-day initial starvation caused shorter shells and lower percentage of competent larvae. Three days of continuous feeding was required for 50% of the larvae to reach competence. After feeding for 3 days, most larvae could become competent to metamorphose even under starvation. The time of starvation was also critical: larvae that suffered 1-day food deprivation in the first 2 days of larval release had shorter shells and lowered percent competent larvae than those that suffered the same length of food deprivation in later stages of development. Our study thus indicates that both food concentration and short-term starvation have detrimental effects on the larval development of this species, and that once the larva has consumed certain amount of food, starvation may induce metamorphosis.     

Local requirement of the Drosophila insulin binding protein imp-L2 in coordinating developmental progression with nutritional conditions

In Drosophila, growth takes place during the larval stages until the formation of the pupa. Starvation delays pupariation to allow prolonged feeding, ensuring that the animal reaches an appropriate size to form a fertile adult. Pupariation is induced by a peak of the steroid hormone ecdysone produced by the prothoracic gland (PG) after larvae have reached a certain body mass. Local downregulation of the insulin/insulin-like growth factor signaling (IIS) activity in the PG interferes with ecdysone production, indicating that IIS activity in the PG couples the nutritional state to development. However, the underlying mechanism is not well understood. In this study we show that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2), a growth inhibitor in Drosophila, is involved in this process. Imp-L2 inhibits the activity of the Drosophila insulin-like peptides by direct binding and is expressed by specific cells in the brain, the ring gland, the gut and the fat body. We demonstrate that Imp-L2 is required to regulate and adapt developmental timing to nutritional conditions by regulating IIS activity in the PG. Increasing Imp-L2 expression at its endogenous sites using an Imp-L2-Gal4 driver delays pupariation, while Imp-L2 mutants exhibit a slight acceleration of development. These effects are strongly enhanced by starvation and are accompanied by massive alterations of ecdysone production resulting most likely from increased Imp-L2 production by neurons directly contacting the PG and not from elevated Imp-L2 levels in the hemolymph. Taken together our results suggest that Imp-L2-expressing neurons sense the nutritional state of Drosophila larvae and coordinate dietary information and ecdysone production to adjust developmental timing under starvation conditions.     

Differential replication of ribosomal DNA during larval development in Calliphora (Diptera)

The hybridization of ribosomal RNA with DNA extracted from whole insects has been compared at six stages during the development of the blowfly Calliphora stygia. Total DNA from hatching larvae contains approximately 20% more rDNA than does total DNA from either mid-embryonic or adult insects. DNA from late third-instar larvae close to pupariation contains about 30% less rDNA than that from hatching larvae. The proportion of rDNA to total DNA in larvae of intermediate ages falls between these two extremes. Ribosomal RNA cistrons are thus replicated more rapidly than the remainder of the genome in the late embryonic or earliest larval stages, but more slowly during the later cycles of DNA synthesis in those larval tissues which develop high levels of polyteny.     

Effects of starvation on larval growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum

The effects of starvation on larval growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum at the temperature of 19.6–21.6 °C, the salinity of 34‰ and pH of 8.0 were investigated from May 18 to July 18, 2006. In this study, the early, middle and late umbo-veliger larvae with the shell lengths of 100, 140, and 190 μm were subject to temporary food deprivation for up to 4.5, 20, and 25d at 0.5, 4, 5d intervals, followed by refeeding for the remaining of a 24, 20, 25d period, respectively. The results suggested that the larvae should have shown considerable tolerance to starvation due to their endogenous and exterior nutrition material, for larvae and time to the point-of-no-return (PNR: the threshold point during starvation after which larvae could no longer metamorphose even if food is provided) were calculated to be 4.25, 17.54, and 22.17d. As the starvation period prolonged, the mean shell length of larvae starved got close to constants at 1.5, 4, and 15d after starvation, which were different for larvae at different stages when starvation began, survival of larvae decreased, and was lower in treatments starved earlier in development than those starved later, for the early, middle and late umbo-veliger larvae, after 4.5, 20 and 25d of starvation period, few larvaes were alive. After starvation period, the alive larvaes were able to metamorphose and had a capability of compensatory growth when refeeding was given. Starvation not only affected metamorphosis rate, but also caused the delay in the time to metamorphosis and the decrease in the metamorphosed sizes. For example, for the continuously-fed larvae, duration to metamorphosis was 20.7d, for larvae with a size of 100-μm starved for up to 4d, larvae with a size of 140-μm starved for up to 16d, larvae with a size of 190-μm starved for up to 20d, duration to metamorphosis were 29.7, 31.7, and 37.7d, the delay in duration to metamorphosis were 9, 11, and 17d, respectively. Furthermore, importance of nutrition material for maintaining larval survival during starvation and the compensatory growth on larvae at the same feeding time were discussed.     

Effects of age and liquid holding on the UV-radiation sensitivities of wild-type and mutant Caenorhabditis elegans dauer larvae

The dauer larva is a facultative developmental stage in the life cycle of the nematode Caenorhabditis elegans. Dauer larvae, which can survive under starvation for over 60 days, resume normal development when feeding is resumed. Wild-type (N2) and 4 radiation-sensitive (rad) mutant dauer larvae were tested for their abilities to develop into adults after UV-irradiation. The rad-3 mutant was over 30 times as sensitive as N2; rad-1, rad-2 and rad-7 mutants were not hypersensitive. Irradiation also delayed development in survivors. Wild-type dauer larvae did not differ in radiation sensitivity from 0 through 50 days of age. There was no liquid holding recovery (LHR); that is, survival did not increase when wild-type dauer larvae were held in buffer after irradiation.     

The contribution of the fatbody to RNA and ribosomal changes during development of the blowfly Calliphora erythrocephala (Meig)

The initiation of blowfly metamorphosis is associated with a pronounced decrease in the number of larval ribosomes; this reduced number then remains constant throughout pharate adult development. Ribosomal RNA accounts for most of the total RNA in larvae shortly after the cessation of feeding and growth, but thereafter the amount of rRNA declines disproportionately to total RNA until early pharate adult development; thereafter, the ratio remains constant until adult emergence. Larval fatbody ribosomes, which constitute about half of the total in the entire organism, are destroyed in situ prior to pupariation. The progressive decrease in fatbody rRNA is accompanied by a corresponding increase in a degraded, relatively insoluble 4–7 S nucleic acid which, stored until adult emergence when it is discarded, accounts for the disparity between total RNA and rRNA. The extracellular ribosomes previously observed during pharate adult development are thus derived from dissolution of larval tissues other than fatbody.     

Haemolymph ecdysteroid titres in diapause- and non-diapause-destined larvae and pupae of Sarcophaga argyrostoma

No differences were observed between the rates of development of larvae and pupae from diapause- and non-diapause-destined lines of Sarcophaga argyrostoma except that those destined for diapause have a longer post-feeding, wandering, larval phase associated with a lower haemolymph ecdysteroid titre, as measured by radioimmunoassay. Following pupariation, both cultures show a high haemolymph titre associated with larval/pupal apolysis. The developing culture displays an ecdysteroid peak at 72 h after pupariation which may be involved with pupal/adult apolysis and the initiation of pharate-adult development. This peak is reduced in the diapause-destined culture. Following the initiation of pharate adult development, there is a very large peak at 85–90 h. Those pupae entering diapause display very low titres as a result of the failure of the brain/prothoracic gland axis to release ecdysone. There are no quantitative or qualitative differences between the titres of specific ecdysteroids in the prepupae of the two lines as determined by reverse-phase high-performance liquid chromatography. A preliminary examination of the levels of free and conjugated ecdysteroids has provided the basis for proposing a mechanism of ecdysone metabolism in this insect.     

Juvenile hormone titres and metabolism during starvation-induced supernumerary larval moulting of the tobacco hornworm, Manduca sexta L.

When tobacco hornworm (manduca sexta) larvae are starved for 5 days immediately after ecdysis to the 5th instar, then fed normal diet, they undergo a supernumerary moult instead of metamorphosis. During starvation the titre of juvenile hormone in the haemolymph increased to a maximum of 3 ng juvenile hormone I equivalents/ml (determined by the black Manduca larval bioassay) on the fourth day of starvation, then began a decline which continued through the subsequent feeding period. The changes in juvenile hormone titre were not attributable to changes in haemolymph volume during starvation (only a 5% decrease) and subsequent feeding. During starvation the esterase activity of the haemolymph declined 4-fold with a 2-fold larger decrease in the DFP-insensitive, presumably juvenile hormone specific, esterase activity. Both the total and the juvenile hormone-specific esterase activity then increased as a function of larval weight during the subsequent feeding period. As growth was slow in the prolongedly starved larvae, sufficient juvenile hormone was present at the time of prothoracicotropic hormone (PTTH) and ecdysteroid release at the beginning of the fourth day of feeding to prevent metamorphosis.     

Reproductive strategies of marine benthic invertebrates revisited: facultative feeding by planktotrophic larvae

Fecundity-time models of reproductive strategies in marine invertebrates all predict that reproductive success is maximized only at the extreme levels of investment. Selection should drive egg sizes toward small eggs and planktotrophy or large eggs and lecithotrophy. The existence of two distinct larval types, feeding and nonfeeding, has been taken as confirmation of this prediction and has established the current paradigm for larval ecology. However, comparative and experimental evidence does not support the prediction that egg size is minimized in species with planktotrophic larvae. Recent discoveries have documented the existence of planktotrophs that have intermediate egg sizes, differing degrees of dependence on exogenous food, and differing capacities for facultative feeding. A fecundity-time model is presented that includes facultative larval feeding by dissociating the onset of feeding capability from the need for exogenous food. The facultative feeding model shows that reproductive success can be maximized at intermediate levels of investment per offspring between the minimum for development and the threshold for lecithotrophy, depending on the amount of food available to larvae and the intensity of planktonic mortality. A continuum of larval strategies is predicted.