The life cycle of the nematode Caenorhabditis elegans: II. A simplified method for mutant characterization

A simple method for characterizing the development and reproduction of mutant strains of the small nematode Caenorhabditis elegans has been developed. “3-egg” populations of nematodes, started with three synchronously laid eggs and allowed to develop undisturbed for about two generations, are measured on the electronic nematode counter, and the resulting size distributions are interpreted by a computer. The computer compares the observed distribution to an expected distribution, generated by assuming the developmental curves previously measured for the wild-type C. elegans; if the distributions do not agree, the computer independently varies scale factors for developmental rate, size, egg-laying rate, and spread until the expected distribution best approximates the observed one. The resulting factors quantify any mutant defect of growth or reproduction, and the poorness of fit tells how greatly the mutant's development differs from that of the wild-type in ways other than those allowed for by the four scale changes. The computer program is shown to be able to fit wild-type C. elegans 3-egg populations grown for various lengths of time at 20°C. Three-egg populations of wild-type animals grown at 16 and 25°C are fitted by the computer and give altered developmental parameters consistent with those previously measured by more direct means. Nine behavioral and morphological mutants have been analyzed by this method. All show some developmental alterations from the wild-type. Fertility seems to be more adversely affected than growth. One mutant has been studied in more detail to determine the specific ways in which it differs from the wild-type.     

Effects of temperature on growth, development and diapause in the yellow dung fly – against all the rules?

The effects of rearing temperature (and photoperiod) on growth, development, body size, and diapause induction and termination in the yellow dung fly, Scathophaga stercoraria, were investigated by allowing replicate families of larvae to develop in the field along a time sequence approaching the onset of winter. This was supplemented with extensive laboratory rearing. At constant laboratory temperatures, growth rates were maximal between 15°C and 20°C and decreased at higher (25°C) and lower (10°C) temperatures, while the development rate was maximal at 25°C. Perhaps related to this, yellow dung flies reached a given size faster at naturally variable, as opposed to constant, temperatures. In the field, lower temperatures towards the end of the season resulted in larger individuals that grew faster. Adult body size increased as development time, expressed in calendar days, increased, a positive relationship commonly taken for granted in life history theory, but decreased as development time expressed in degree-days increased. The effect of temperature on growth, development and body size can thus change or even reverse if individuals can alter their growth rate independently of development time, and if the physiological effects of temperature are factored out by converting development time into degree-days above a lower development threshold. Therefore, supposedly well-established trends possibly need to be re-examined along these lines. Pupal winter diapause towards the end of the season was highly reversible by temperature. Pre- and post-winter emergence patterns together suggest that the minimum time for yellow dung flies to successfully complete development, at any time of the year, is about 230–250 degree-days. Received: 2 September 1996 / Accepted: 26 February 1997     

The effect of salinity and temperature on the larval development of clibanarius vittatus (Bosc) (crustacea: decapoda: diogenidae)

Clibanarius vitatus (Bosc) larvae were reared in twenty combinations of four salinities (15, 20, 25, and 30%) and five temperatures (15, 20, 25, 30 and 35%°C). No development was observed in any salinity at 15°C, but partial development occurred in all other test conditions. Metamorphosis to juvenile crabs was noted only at salinities of 25 and 30A% in combination with temperatures of 25 and 30°C. In general, development times were decreased at higher temperatures; no trend was evidence for salinity. Mortality of zoeae was usually highest at the time of the first molt and greatest overall mortality occurred during the megalopa stage prior to metamorphosis. Previous experiments (unpubl.) have shown that C. vittatus adults can tolerate temperature down to 5°C. It is suggested that geographic distribution of C. vittatus (Virginia, southward) is limited not by adult tolerances but by the inability iof the species to establish a breeding population. Larvae require two months at 25°C or above to metamorphose, and this condition is not met in areas north of Virginia.     

Metarhizium spp. isolates from madagascar: Morphology and effect of high temperature on growth and infectivity to the migratory locust,Locusta migratoria

Five strains ofMetarhizium anisopliae (Metsch.) Sorokin and one strain ofMetarhizium flavoviride Gams &; Rozsypal originally isolated in Madagascar were studied. Measurements of conidia and, for the first time, also of blastospores produced in a liquid medium were used for species and variety determination. Blastospores ofM. flavoviride were more homogenous in their size than those ofM. anisopliae. Growth at high temperatures between 25° and 40°C showed that 4 isolates ofM. anisopliae grew at 36°C andM. flavoviride grew at 38°C. Using alternating day/night temperatures (8/16 h) the three strains tested could also tolerate 40°/25°C. In bioassays, fiveMetarhizium spp. isolates were tested against third and fourth instar larvae ofLocusta migratoria (L.) at two alternating day/night temperatures of 30°/25°C and 36°/25°C. In the cooler regime, all strains caused a mortality of 50% within 5.9 to 8.5 days (median lethal time), while in the 36°/25°C treatment only the thermophilicM. flavoviride and oneM. anisopliae strain isolated from a soil sample gave comparable results with median lethal times of 6.8 and 7.3 days, respectively.     

Effects of temperature on growth and efficiency of food utilization in fifth-instar caterpillars of the tobacco hornworm, Manduca sexta

Fifth-instar larvae of Manduca sexta were reared from hatching on artificial diet at 15, 20, 25, 30 and 35°C. Total development time decreased with increasing temperature. Very few larvae (12%) survived at 15°C, so this temperature was not considered further. There was some mortality at 30°C (11%), and at 35°C (50%).The absolute rate of growth in the fifth instar was faster at 25 than at 20°C, but was similar at 25, 30 and 35°C. This was true both for caterpillars that were chronically exposed to experimental temperatures (i.e. since hatching) and for those acutely exposed (i.e. reared up to fifth instar at 25°C).There was a progressive decrease with higher rearing temperatures in both the initial and final sizes of chronically exposed fifth-instar larvae. Acutely exposed caterpillars matched for initial size showed smaller temperature related differences in final size. Because of these size differences there were differences in relative growth rate which did not reflect true differences in absolute growth rate.Total food consumed by chronically exposed caterpillars was greatest at the lowest temperature (20°C), and decreased progressively with increasing temperature. The absolute rate of food consumption increased from 20 to 25°C, but did not vary significantly between 25 and 35°C. Differences in the sizes of the insects at the different temperatures meant that there were differences among relative measures of consumption that did not reflect absolute food consumption.For chronically exposed caterpillars, none of the three usual indices of food conversion efficiency (AD, ECI and ECD) varied significantly with temperature between 20 and 35°C. This implies that the effects of temperature on metabolic costs are closely matched to food consumption.Oxygen consumption increased with temperature between 20 and 25°C but was temperature compensated between 25 and 35°C.These findings are discussed in terms of their implications for the optimal temperature for growth in Manduca.     

BERGMANN SIZE CLINES: A SIMPLE EXPLANATION FOR THEIR OCCURRENCE IN ECTOTHERMS

In general ectothermic organisms grow larger at both lower temperatures and higher latitudes. Adult size in the soil nematode Caenorhabditis elegans reared at 10°C was approximately 33% greater than worms grown at 25°C. Nematode egg size and fish red blood cell size showed similar size increases at lower temperatures. These results indicate that body size differences in many ectotherms may simply be a consequence of developmental processes that cause cells to grow larger at lower temperatures. This would provide a general explanation for the increased size of ectotherms at lower temperatures independent of species-specific ecology.     

Phocanema decipiens: Molting in seals

Infective larvae of the anisakine nematode Phocanema decipiens from the muscle of Atlantic cod (Gadus morhua) were fed to harbor seals (Phoca vitulina) and gray seals (Halichoerus grypus). During maturation in the stomach of seal hosts, P. decipiens molted twice; these molts are the third and fourth of its life cycle. The third molt occurred between the second and fifth days of infection. The third stage, i.e., infective larva entering the third molt, had a cuticular tooth ventral to the mouth; the fourth stage larva emerging from the third molt had three bilobed lips with dentigerous ridges. The fourth molt occurred between the 5th and 15th days in seals. A female nematode emerging from the fourth molt possesses a vulva and a vagina; a male possesses caudal alae, pre- and postanal papillae. Significant morphometric changes in nematodes were associated with both molts. Females and males of P. decipiens reached maturity after 15 to 25 days in seals. Ova were detected in the feces of the seal hosts as early as the 16th day.     

Factors influencing the pathogenicity and development of Mattesia trogodermae infecting Trogoderma glabrum larvae

Factors that regulate development of Mattesia trogodermae in Trogoderma glabrum were defined, and their quantitative effects were determined. The rate of and the extent to which spore formation proceeds is strictly governed by temperature. More spores are produced at 30° than at 25°C and very low numbers of spores are formed when the incubation temperature is 35°C. When insects are incubated at 35°C for 1–10 days and transferred to 30°C for the remainder of the 30-day experiment, spore production capacity gradually declines with increasing time at 35°C. Two hypotheses are proposed for this phenomenon. Larval size also regulates the extent of spore production, larger larvae having greater potential for spore development. This is not influenced by dosage. Spore production in pupae and adults was always retarded.Dosage and environmental conditions which influence the virulence of M. trogodermae were investigated. These studies show that rates of mortality are higher at higher temperatures. Low doses of spores result in longer LT50's than do high doses at 25° and 30°C. No differences in rates of mortality were found between different doses at 35°C.     

Pathogenicity of Criconemella curvata to Alfalfa

Large populations of Criconemella curvata and extended experimental periods were required to adversely affect yield of ''Moapa 69'' alfalfa. C. curvata ectoparasitic feeding caused a reduction in feeder root numbers and tap root size and small lesions on tap and secondary roots. Greater reproduction occurred at 27 C than at 22 C, but the effect of the nematode on alfalfa growth was the same at both temperatures.     

The life history of Labiostrongylus eugenii, a nematode parasite of the Kangaroo Island wallaby (Macropus eugenii): development and hatching of the egg and the freeliving stages

Smales L. R. The life history of Labiostrongylus eugenii, a nematode parasite of the Kangaroo Island Wallaby (Macropus eugenii): development and hatching of the egg and the free living stages. International Journal for Parasitology7: 449–456. Labiostrongylus eugenii (Trichonematidae) occurs in the stomach of the Kangaroo Island Wallaby. Egg morphology is similar to that of other strongyloids. When incubated at 25°C embryogenesis is completed in about 30 h. An incomplete moult occurs within the egg, and larvae hatch at a sheathed second-stage $\text{43}\text{1}\text{2}\text{–83}\text{1}\text{2}$ h later. Development occurred at all temperatures between 2° and 37°C with an optimum about 25°C and an upper limit near 37°C. The hatching process is very rapid, taking about 2 min. It is signalled by increased larval activity followed by a change in shell permeability. The larva hatches at that pole of the shell which has become plastic.The sheathed second-stage larva measures 659.50 ± 22.54 μm by 27.98 ± 1.22 μm. Its internal structures are concealed by a mass of opaque granules which were demonstrated as neutral lipid by oil red O staining. A second incomplete moult at 3–4 days results in a doubly sheathed infective larva from which the lipid gradually disappears. The mouth never appears patent and the larvae neither feed nor grow but rather decrease in size with age. Optimal temperatures for larvae range between 15°–25°C with 37°C about the upper limit. The significance of this developmental pattern is discussed.     

Thermal limits for the establishment and growth of populations of the invasive apple snail <Emphasis Type="Italic">Pomacea canaliculata</Emphasis>

Pomacea canaliculata is a South American freshwater snail considered as one of the world’s worst invasive alien species. A temperature of around 25 °C has usually been considered to be optimal for rearing P. canaliculata. Nevertheless, snails have not been reared under a wide range of temperatures to reveal the optimum for performance in terms of population increase. We investigated the effect of temperature on growth, survival and reproduction, estimating demographic parameters for P. canaliculata in the wide range of temperatures at which these snails are active (15–35 °C). No reproductive activity was evidenced for the snails reared at 15 °C, probably explained by the small sizes attained at this temperature. Temperatures above 25 °C did not promote a significant acceleration in growth so higher temperatures will not result in a reduction in time to reach maturity. In fact, snails from 25 and 30 °C began reproduction at the same age. We report here for the first time a detrimental effect of high temperatures that provoked a significant decrease in the contribution of snails to the next generation: the viability of eggs from the snails reared at 30 °C was very low and the snails exposed to a constant water temperature of 35 °C were unable to produce eggs. Our findings reveal a new environmental constraint that could be a determinant of the range limits of this species in invaded regions, especially during the coming decades, anticipating the scenario predicted from global warming.     

Interactions between Escherichia coli and the plant‐parasitic nematode Meloidogyne javanica

Aims: To determine the potential of the plant‐parasitic nematode Meloidogyne javanica to serve as a temporary reservoir for Escherichia coli. Methods and Results: The adhesion to and persistence of E. coli on the surface of M. javanica were evaluated at different times and temperatures. A pure culture of green fluorescent protein (GFP) tagged E. coli was mixed with ca. 1000 J2 M. javanica for 2 h at 25°C. The nematodes were then washed and the rate of the adhesion of the bacteria to the nematodes was determined by counting the viable nematode‐associated E. coli, and by fluorescence microscopy. A dose‐dependent adhesion rate was observed only at a bacterium to nematode ratio of 10⁴–10⁶ : 1. The adhesion of E. coli to the nematodes was also tested over a 24 h‐period at 4°C, 25°C and 37°C. At 4°C and 37°C, maximal adhesion was observed at 5 h; whereas at 25°C, maximal adherence was observed at 8 h. Survival experiments showed that the bacteria could be detected on the nematodes for up to 2 weeks when incubated at 4°C and 25°C, but not at 37°C. Conclusions: Under laboratory conditions, at 4°C and 25°C, M. javanica could serve as a temporary vector for E. coli for up to 2 weeks. Significance and Impact of the Study: These findings support the hypothesis that, in the presence of high concentrations of E. coli, M. javanica might serve as a potential vehicle for the transmission of food‐borne pathogens.     

Effect of temperature on germination,growth, and infectivity of the mosquito pathogen Tolypocladium cylindrosporum (deuteromycotina; hyphomycetes)

The mosquito pathogen Tolypocladium cylindrosporum was examined with regard to its response to temperature. Similar temperature ranges were found for growth, germination, and infectivity of blastospores and conidia. Germination occurred at 8° and 33°C but not at 6° and 35°C. Optimal germination and growth was noted between 24° and 27°C for both spore types. Infectivity of blastospores and conidia at different temperatures was examined by exposing L₂Aedes sierrensis larvae to concentrations of 5 × 10⁵ blastospores/ml or 5 × 10⁶ conidia/ml. Larvae were incubated at 12°, 15°, 25°, and 30°C. Infection occurred at all temperatures tested with LT₅₀ values ranging from 22.7 days (12°C) to 5.6 (25°C) days for conidia and 4.7 days (12°C) to 0.6 day (25°C) for blastospores. These results confirmed earlier findings that blastospores infected and killed host larvae more rapidly than conidia and suggested that this difference is largely due to the more rapid germination rate of blastospores. These experiments demonstrated that T. cylindrosporum can be active against mosquito larvae over a broad range of temperatures encompassing both the cold-water habitat of certain temperate mosquito species as well as the habitat of tropical vector species.     

The influence of temperature variations and thermal pollution on various aspects of the biology of the prawn Palaemon pacificus (Stimpson)

The influence of different temperatures 10, 15, 20, and 25°C on the food consumption, growth, moulting rate, and respiration of Palaemon pacificus (Stimpson) from Langebaan Lagoon, west coast of South Africa, was studied under laboratory conditions. At 10°C mortality was high so that food consumption and moulting rate could not be determined as these were very low. At higher temperatures, food consumption was found to be temperature dependent, the rate at 25°C being twice that at 15°C. Growth rate was most favourable at 25°C. At 28°C growth rate was lower than at 20°C but higher than at 15°C. The intermoult period was 17 days at 15°C, and 11 and 10 days at 20, and 25°C, respectively. It seems that from an energetic point of view, 25°C is the most favourable temperature for P. pacificus. Several indices of growth efficiency at different temperatures are presented. The appearance of this prawn in South African west coast localities such as Langebaan during the summer and its disappearance during winter, can be explained by its temperature preferences. The possibility that thermal pollution from a nuclear power station may be beneficial to this prawn, is discussed.     

Dauer Larvae of Caenorhabditis briggsae in Axenic Culture

The free-living hermaphroditic nematode, Caenorhabditis briggsae, enters a dauer stage under certain conditions in axenic culture. Dauer larvae differ from directly-developing third-stage larvae in internal structure, size at time of second molt, morphology of second and third cuticles, separation zone of cuticular caps, and survival at 4 C and 37 C, temperatures fatal to other stages. Males, which occur rarely in liquid medium, may mature under conditions which cause most of the hermaphrodites to go into the dauer stage, resulting in a culture with increased male-to-hermaphrodite ratio.     

The effect of temperature on energy distribution during the last-larval stadium of the female house cricket, Acheta domesticus

The distribution of energy during the last stadium of the house cricket at two temperatures was the main theme of this study. Food consumption, growth, and oxygen consumption were greater in the first half of the stadium at both 25 and 35°C. An RQ > 1 indicated the conversion of carbohydrates to lipids during the first half of the instar at both temperatures. The duration of the stadium increased from 6 days at 35°C to 14 days at 25°C. The same maximal weight, protein content and lipid content were attained at both 25 and 35°C. A weight loss (mostly in stored lipids) after the midstadium peak weight was greater at the lower temperature. The absorption efficiency and the production of metabolic wastes were not affected by temperature, but the metabolic efficiency was much higher at 35 than at 25°C during the first half as well as the latter half of the stadium. Although during the first half of the stadium more energy was ingested, absorbed, and made available for growth at 25 than at 35°C, only slightly more growth occurred at 25°C. During the last half of the stadium less energy was ingested at 25 than at 35°C, and much more growth occurred at 35°C because of the even greater heat loss at 25 than at 35°C. Therefore at a lower temperature cricket larvae eat slightly more and reach the same maximal weight as at a higher temperature, but they end up smaller because they waste more energy during the extended duration of the stadium at the lower temperature.     

WITHIN- AND BETWEEN-GENERATION EFFECTS OF TEMPERATURE ON THE MORPHOLOGY AND PHYSIOLOGY OF DROSOPHILA MELANOGASTER

We investigated the effects of developmental and parental temperatures on several physiological and morphological traits of adult Drosophila melanogaster. Flies for the parental generation were raised at either low or moderate temperature (18°C or 25°C) and then mated in the four possible sex-by-parental temperature crosses. Their offspring were raised at either 18°C or 25°C and then scored as adults for morphological (dry body mass, wing size, and abdominal melanization [females only]), physiological (knock-down temperature, and thermal dependence of walking speed), and life history (egg size) traits. The experiment was replicated, and the factorial design allows us to determine whether and how paternal, maternal, and developmental temperatures (as well as offspring sex) influence the various traits. Sex and developmental temperature had major effects on all traits. Females had larger bodies and wings, higher knock-down temperatures, and slower speeds (but similar shaped performance curves) than males. Development at 25°C (versus at 18°C) increased knock-down temperature, increased maximal speed and thermal performance breadth, decreased the optimal temperature for walking, decreased body mass and wing size, reduced abdominal melanization, and reduced egg size. Parental temperatures influenced a few traits, but the effects were generally small relative to those of sex or developmental temperature. Flies whose mother had been raised at 25°C (versus at 18°C) had slightly higher knock-down temperature and smaller body mass. Flies whose father had been raised at 25°C had relatively longer wings. The effects of paternal, maternal, and developmental temperatures sometimes differed in direction. The existence of significant within- and between-generation effects suggests that comparative studies need to standardize thermal environments for at least two generations, that attempts to estimate “field” heritabilities may be unreliable for some traits, and that predictions of short-term evolutionary responses to selection will be difficult.     

Some factors affecting survival and oxygen uptake in a subtropical beach flea

The survival and oxygen uptake of the supralittoral amphipod Chroestia lota Marsden & Fenwick were investigated in humid air and sea water between 15 and 35°C. Seven-day exposure experiments were made on three size groups of amphipods at 6 constant temperatures (15, 20, 25, 30, 35, 40 °C) and three cyclic temperatures (15–25, 20–30, 25–35°C) in air and in sea water at 34 and 17%. salinity. Neither size, treatment nor temperature affected survival between 15 and 30°C. Mortality increased > 30°C with large individuals being consistently less tolerant than medium and small amphipods. While amphipods exposed to cyclic temperatures during submersion had reduced survival compared with constant temperatures, those individuals exposed to cyclic conditions in humid air showed the greatest resistance. Oxygen uptake of Chroestia increased with dry body wt and, over the range 15–35°C, this semi-terrestrial beach flea could maintain its aerial VO₂ following submersion. Oxygen uptake increased directly in proportion to gill area and the weight specific gill area was low, consistent with the need to reduce desiccation. It is suggested that total gill area does not limit oxygen uptake in Chroestia and that cutaneous respiration may be important especially in aquatic conditions.     

Temperature response of root‐lesion nematode (Pratylenchus thornei) reproduction on wheat cultivars has implications for resistance screening and wheat production

The root‐lesion nematode Pratylenchus thornei is a major pathogen of wheat and other field crops, particularly in the northern grain region of sub‐tropical eastern Australia. Research was conducted into the temperature requirements of P. thornei for reproduction on wheat to increase the reliability of selection in resistance tests for wheat breeding. Final population densities (P_f) of P. thornei were determined on four wheat cultivars (Gatcher, GS50a, Potam and Suneca) at fortnightly intervals from 8 to 18 weeks at a range of six soil temperatures (15°C, 20°C, 22.5°C, 25°C, 27.5°C and 30°C) in a glasshouse experiment. Pratylenchus thornei had the highest P_f in the temperature range of 20–25°C on all wheat cultivars at all growth times after sowing, with no nematode reproduction measured at 30°C and very little at 15°C. The wheat cv. GS50a consistently produced lower P_f than cvs Gatcher, Potam and Suneca in the optimum temperature range of 20–25°C. In carrot disc cultures, P. thornei had an optimum temperature of 25°C with little reproduction at 17.5°C and none detectable at 30°C. A standard soil temperature of 23°C was chosen to maximise differences in nematode reproduction between resistant and susceptible wheat genotypes for selection in wheat breeding, and to improve reproducibility among successive experiments. The relationships derived from these experiments will be valuable for simulation of P. thornei reproduction in crop growth models. They also indicate that early sowing of wheat into cool soil (≤15°C) in farmers' fields of the northern grain region should favour wheat growth over nematode reproduction and increase grain yield.     

Effects of ecdysteroids on reproductive physiology of Nippostrongylus brasiliensis (nematoda) in vivo

1. The influence of ecdysteroids (ecdysone and ecdysterone) was investigated on the control of reproduction in the parasitic nematode Nippostrongylus brasiliensis in vivo.2. Infestive larvae (L₃) were immersed in solutions of ecdysteroids (2.2 μM) at 37°C for 4 hr before injection into the host. The effect on egg-laying was observed two stages later.3. The treatment increased egg-laying, but had no influence on the timing of the reproductive period. The greatest effect was observed with ecdysone, ecdysterone only inducing a small and non-significant stimulation under our experimental conditions.4. The physiological role of ecdysteroids in meiosis and gonadal development in nematodes is discussed.