Changes in lipid contents of infective third-stage larvae of Necator americanus during desiccation and revival

Changes in lipid content of infective third-stage larvae of Necator americanus were investigated after short periods of induced desiccation and revival. A fall in lipid reserve from an outset level of 86% to 74% was recorded in the first 2 h of desiccation. With increased desiccation, lipid reserves did not show significant decline, probably as a result of decreased lipid metabolism in the desiccated larvae. During revival, there was a drastic fall in lipid reserves as a result of increased lipid utilisation by the reviving larvae. The results showed that desiccated larvae with lipid levels less than 10% did not revive. The presence of lipid did not appear to prevent desiccation but was an essential factor for revival. The ecological significance of these findings in field larvae is discussed.     

Desiccation resistance in pre-diapause, diapause and post-diapause larvae of Choristoneura fumiferana (Lepidoptera: Tortricidae)

Desiccation resistance was examined in pre-diapause, diapause and post-diapause larvae of the spruce budworm, Choristoneura fumiferana (Clemens), in terms of passive water evaporation under three desiccation conditions: freeze-drying, desiccant-drying at 2 degrees C and desiccant-drying at 18 degrees C. Diapausing second instar larvae and post-diapause non-feeding second instar larvae showed strongest desiccation resistance: a significant amount of water was retained after repeated drying under desiccating conditions, while pre-diapause first instar larvae and post-diapause feeding second instar larvae lost almost all their water content after one or two drying cycles. A hibernaculum covering had no effect on water evaporation. While dead larvae tended to lose significantly more water than their living counterparts, particularly among first instar larvae, such an impact was much weaker among diapausing second instar larvae. Desiccation resistance was lost when post-diapause second instar larvae were allowed access to water while the level of desiccation resistance was maintained or enhanced when the larvae did not have access to water. These results are discussed in the context of overwintering ecology of the species and possible mechanisms for the desiccation resistance are also discussed.     

Necator americanus: temperature, pH, light, and larval development, longevity, and desiccation tolerance

The effect of incubation temperature and pH on the hatch rate of eggs of Necator americanus, and the desiccation tolerance of the resulting infective stage-3 larvae were investigated in the laboratory under controlled conditions. Hatching did not occur below 15 C and above 35 C. A 21% hatch rate was obtained at 15 C while a 10.6% hatch rate was obtained at 35 C. The highest hatch rate (93.7%) was obtained at 30 C. The optimum pH for hatching was 6.0, but the larvae did not reach the infective stage. Incubation temperature of the eggs affected the longevity and desiccation tolerance of resultant infective larvae. Larvae hatched at 30 C and maintained at 26 C under bright fluorescent light had a 50% survival time (S50) of 4 days. In the dark or shade, the S50 for larvae raised at 30 C was 5 weeks, while that of larvae hatched at 20 C was 7 weeks. Incubation temperature also affected the desiccation tolerance of larvae. Larvae developed at 20 C were more resistant to desiccation at various relative humidity values than larvae hatched at 30 C.     

Identification of a gene, Desiccate, contributing to desiccation resistance in Drosophila melanogaster

Suitable alterations in gene expression are believed to allow animals to survive drastic changes in environmental conditions. Drosophila melanogaster larvae cease eating and exit moist food to search for dry pupation sites after the foraging stage in what is known as the wandering stage. Although the behavioral change from foraging to wandering causes desiccation stress, the mechanism by which Drosophila larvae protect themselves from desiccation remains obscure. Here, we identified a gene, CG14686 (designated as Desiccate (Desi)), whose expression was elevated during the wandering stage. The Desi expression level was reversibly decreased by transferring wandering larvae to wet conditions and increased again by transferring them to dry conditions. Elevation of Desi expression was also observed in foraging larvae when they were placed in dry conditions. Desi encoded a 261-amino acid single-pass transmembrane protein with notable motifs, such as SH2 and PDZ domain-binding motifs and a cAMP-dependent protein kinase phosphorylation motif, in the cytoplasmic region, and its expression was observed mainly in the epidermal cells of the larval integuments. Overexpression of Desi slightly increased the larval resistance to desiccation stress during the second instar. Furthermore, Desi RNAi larvae lost more weight under dry conditions, and subsequently, their mortalities significantly increased compared with control larvae. Under dry conditions, consumption of carbohydrate was much higher in Desi RNAi larvae than control larvae. Based on these results, it is reasonable to conclude that Desi contributes to the resistance of Drosophila larvae to desiccation stress.     

Effects of the desiccation on Biomphalaria tenagophila (Orbigny, 1835) (Mollusca) infected by Schistosoma mansoni Sambon, 1907

Specimens of Biomphalaria tenagophila exposed to miracidia of Schistosoma mansoni were submitted to different desiccation periods as follows: group I: 24 h after exposure, desiccated for 28 days; group II: after cercariae elimination, desiccated for 7 days; group III: 21 days after exposure, desiccated for 7 days; group IV: 14 days after exposure, desiccated for 14 days; group V: 7 days after exposure, desiccated for 21 days. From the obtained data it was verified that desiccation was not capable of interrupting the development of larvae of S. mansoni in mollusks. A delay in the development of S. mansoni larvae in groups I, III, IV and V was observed. A pause was verified in the development of S. mansoni larvae in groups II, III, IV and V. Some larvae, in groups I, III, IV and V, did not suffer as a result of desiccation and continued their development. Larvae in the cercariae stage were shown to be more sensitive to desiccation. It was possible to obtain clearing of mollusks infected by sporocysts II and cercariae using a period of 7 days of desiccation.     

Divergence of larval resource acquisition for water conservation and starvation resistance in Drosophila melanogaster

Laboratory selection experiments have evidenced storage of energy metabolites in adult flies of desiccation and starvation resistant strains of D. melanogaster but resource acquisition during larval stages has received lesser attention. For wild populations of D. melanogaster, it is not clear whether larvae acquire similar or different energy metabolites for desiccation and starvation resistance. We tested the hypothesis whether larval acquisition of energy metabolites is consistent with divergence of desiccation and starvation resistance in darker and lighter isofemale lines of D. melanogaster. Our results are interesting in several respects. First, we found contrasting patterns of larval resource acquisition, i.e., accumulation of higher carbohydrates during 3rd instar larval stage of darker flies versus higher levels of triglycerides in 1st and 2nd larval instars of lighter flies. Second, 3rd instar larvae of darker flies showed ~40?h longer duration of development at 21°C; and greater accumulation of carbohydrates (trehalose and glycogen) in fed larvae as compared with larvae non-fed after 150?h of egg laying. Third, darker isofemale lines have shown significant increase in total water content (18%); hemolymph (86%) and dehydration tolerance (11%) as compared to lighter isofemale lines. Loss of hemolymph water under desiccation stress until death was significantly higher in darker as compared to lighter isofemale lines but tissue water loss was similar. Fourth, for larvae of darker flies, about 65% energy content is contributed by carbohydrates for conferring greater desiccation resistance while the larvae of lighter flies acquire 2/3 energy from lipids for sustaining starvation resistance; and such energy differences persist in the newly eclosed flies. Thus, larval stages of wild-caught darker and lighter flies have evolved independent physiological processes for the accumulation of energy metabolites to cope with desiccation or starvation stress.     

Interactions between desiccation resistance,host-plant contact and the thermal biology of a leaf-dwelling sub-antarctic caterpillar,Embryonopsis halticella (Lepidoptera: Yponomeutidae)

During May 1997 thermal tolerance, supercooling point (SCP), low and high temperature survival, and desiccation resistance were examined in field-fresh Embryonopsis halticella Eaton larvae from Marion Island. SCPs were also examined in acclimated larvae, larvae starved for seven days, larvae within their leaf mines, and in larvae exposed to ice crystals. Field-fresh larvae had a critical minimum temperature (CT(Min)) and critical maximum temperature (CT(Max)) of 0 degrees C and 39.7 degrees C, respectively. Mean SCP of field-fresh caterpillars was -20.5 degrees C and this did not change with starvation. Field-fresh larvae did not survive freezing and their lower lethal temperatures (70% mortality below -21 degrees C) and survival of exposure to constant low temperatures (100% mortality after 12hrs at -19 degrees C) indicated that they are moderately chill tolerant. SCP frequency distributions were unimodal for field-fresh larvae, but became bimodal at higher acclimation temperatures. Contact with ice-crystals caused an increase in SCP (-6.5 degrees C), but contact with the host plant had less of an effect at higher subzero temperatures. It appears that the remarkable desiccation resistance of the larvae is selected for by the absence of a boundary layer surrounding their host plant, caused by constant high winds. This suggests that the low SCPs of E. halticella larvae may have evolved as a consequence of pronounced desiccation resistance.     

Effects of dehydration rate on physiological responses and survival after rehydration in larvae of the anhydrobiotic chironomid

Strategies to combat desiccation are critical for organisms living in arid and semi-arid areas. Larvae of the Australian chironomid Paraborniella tonnoiri resist desiccation by reducing water loss. In contrast, larvae of the African species Polypedilum vanderplanki can withstand almost complete dehydration, referred to as anhydrobiosis. For successful anhydrobiosis, the dehydration rate of P. vanderplanki larvae has to be controlled. Here, we desiccated larvae by exposing them to different drying regimes, each progressing from high to low relative humidity, and examined survival after rehydration. In larvae of P. vanderplanki, reactions following desiccation can be categorized as follows: (I) no recovery at all (direct death), (II) dying by unrepairable damages after rehydration (delayed death), and (III) full recovery (successful anhydrobiosis). Initial conditions of desiccation severely affected survival following rehydration, i.e. P. vanderplanki preferred 100% relative humidity where body water content decreased slightly. In subsequent conditions, unfavorable dehydration rate, such as more than 0.7 mg water lost per day, resulted in markedly decreased survival rate of rehydrated larvae. Slow dehydration may be required for the synthesis and distribution of essential molecules for anhydrobiosis. Larvae desiccated at or above maximum tolerable rates sometimes showed temporary recovery but died soon after.     

Factors Inducing Successful Anhydrobiosis in the African Chironomid Polypedilum vanderplanki: Significance of the Larval Tubular Nest

The African chironomid Polypedilum vanderplanki exhibits anhydrobiosis,i.e., the larvae can survive complete desiccation. Recoveryrate and trehalose content were investigated in larvae desiccatedslowly or at a rate more than 3 times faster. Upon slow desiccation(evaporation rate 0.22 ml day^–1) larvae synthesized 38µg trehalose/individual before complete desiccation, andall of them recovered after rehydration, whereas larvae thatwere dehydrated quickly (evaporation rate 0.75 ml day^–1)accumulated only 6.8 µg trehalose/individual and noneof them revived after rehydration. In the pools that are theirnatural habitat P. vanderplanki larvae make tubes by incorporatingdetritus or soil with their sticky saliva. This tubular structureis a physical barrier not only to protect the larva from naturalenemies but also induces successful anhydrobiosis by reducingthe dehydration rate. When larvae were dehydrated with 100 µldistilled water (DW) in soil tubes, they accumulated 37 µgtrehalose/individual and more than half of them could reviveafter rehydration, whereas larvae without tubes accumulatedlower level of trehalose and none recovered after rehydration.     

Quantitative imaging of Caenorhabditis elegans dauer larvae during cryptobiotic transition

Upon starvation or overcrowding, the nematode Caenorhabditis elegans enters diapause by forming a dauer larva, which can then further survive harsh desiccation in an anhydrobiotic state. We have previously identified the genetic and biochemical pathways essential for survival—but without detailed knowledge of their material properties, the mechanistic understanding of this intriguing phenomenon remains incomplete. Here we employed optical diffraction tomography (ODT) to quantitatively assess the internal mass density distribution of living larvae in the reproductive and diapause stages. ODT revealed that the properties of the dauer larvae undergo a dramatic transition upon harsh desiccation. Moreover, mutants that are sensitive to desiccation displayed structural abnormalities in the anhydrobiotic stage that could not be observed by conventional microscopy. Our advance opens a door to quantitatively assessing the transitions in material properties and structure necessary to fully understand an organism on the verge of life and death.     

Anhydrobiosis increases survival of trichostrongyle nematodes

This study demonstrates that infective-stage larvae of 2 trichostrongyle ruminant gastrointestinal nematodes, Haemonchus contortus and Trichostrongylus colubriformis, can enter into anhydrobiotic states when completely desiccated. Larvae of control trichostrongyle species, Heligmosomoides polygyrus and Nippostrongylus brasiliensis, that infect mice were unable to survive desiccation or to enter into anhydrobiosis. Ruminant larvae were able to survive up to 7 desiccation/rehydration cycles, and, during anhydrobiosis, metabolic activity was decreased and survival of the larvae was prolonged both in the laboratory and in the field. Relative humidity had no effect on ruminant larval survival after anhydrobiosis compared with controls. Temperature had a significant effect, 85.8 +/- 2.3% of larvae in anhydrobiosis could survive low temperatures (0 C) that killed all control larvae. Metabolic activity, measured by changes in lipid content and CO2 respiration, was significantly lower in larvae that entered anhydrobiosis compared with controls (P < 0.05). In field experiments using open-meshed chambers under ambient environmental conditions, larvae in anhydrobiosis had significantly higher survival rates in the field compared with controls (P < 0.05) during summer and winter trials. These data suggest that anhydrobiosis in ruminant larvae promotes survival at freezing temperatures, decreases metabolic activity, and prolongs survival under natural field conditions.     

The limits of drought-induced rapid cold-hardening: Extremely brief,mild desiccation triggers enhanced freeze-tolerance in Eurosta solidaginis larvae

Rapid cold-hardening (RCH) is a highly conserved response in insects that induces physiological changes within minutes to hours of exposure to low temperature and provides protection from chilling injury. Recently, a similar response, termed drought-induced RCH, was described following as little as 6 h of desiccation, producing a loss of less than 10% of fresh mass. In this study, we investigated the limits and mechanisms of this response in larvae of the goldenrod gall fly Eurosta solidaginis (Diptera, Tephritidae). The cold-hardiness of larvae increased markedly after as few as 2 h of desiccation and a loss of less than 1% fresh mass, as organismal survival increased from 8% to 41% following exposure to −18 °C. Tissue-level effects of desiccation were observed within 1 h, as 87% of midgut cells from desiccated larvae remained viable following freezing compared to 57% of controls. We also demonstrated that drought-induced RCH occurs independently of neuroendocrine input, as midgut tissue desiccated ex vivo displayed improved freeze-tolerance relative to control tissue (78–11% survival, respectively). Finally, though there was an increase in hemolymph osmolality beyond the expected effects of the osmo-concentration of solutes during dehydration, we determined that this increase was not due to the synthesis of glycerol, glucose, sorbitol, or trehalose. Our results indicate that E. solidaginis larvae are extremely sensitive to desiccation, which is a triggering mechanism for one or more physiological pathways that confer enhanced freeze-tolerance.     

食物和温湿度对二点委夜蛾幼虫取食和为害玉米苗的影响

【目的】调查显示,二点委夜蛾Athetis lepigone在田间的虫口密度与玉米被害率之间无明显的相关性,田间虫口密度并不能准确预测玉米苗被害率。为了探索二点委夜蛾幼虫发生量与玉米苗被害程度之间的关系,解释田间调查遇到的幼虫即使有时大量发生玉米苗却受损轻微的现象,本研究室内检测了外部环境条件如温度和湿度对二点委夜蛾幼虫取食为害玉米的影响以及昆虫自身的相关取食习性。【方法】将3龄末、4龄初二点委夜蛾幼虫分别置于下述8种温湿度和食物组合条件下进行处理：低温低湿无食物(15℃, 30%RH, 饥饿)、低温干燥无食物(15℃, 0%RH, 饥饿)、低温低湿有食物［15℃, 30%RH, 饲喂人工饲料(AD)］、低温干燥有食物(15℃, 0%RH, AD)、高温高湿有食物(36℃, 90%RH, AD)、高温干燥有食物(36℃, 0%RH, AD)、高温高湿无食物(36℃, 90%RH, 饥饿)、高温干燥无食物(36℃, 0%RH, 饥饿);处理4 h后,评价其对盆栽玉米苗的为害程度,并测定幼虫在4个不同温度(15℃, 24℃, 28℃和33℃)下对玉米叶片的取食量。分别用8种食物(马齿苋、紫苏、甘薯、白菜、大豆、南瓜和玉米叶叶片以及麦秸)将初孵幼虫驯化饲喂至3龄后,检测经过驯化处理后的幼虫对初始驯化食物、麦秸和和玉米叶片的选择性。【结果】在干燥条件下,无论是否经过饥饿处理,幼虫对玉米苗的为害程度均较高,尤其是高温干燥处理后,对玉米苗的平均为害级别达3.1级;低温干燥处理组和高温干燥处理组为害级别在4级以上(含4级)的幼苗数分别占幼苗总数的50%和40%。在15-33℃,二点委夜蛾幼虫的取食量随温度的升高而逐渐增加,48 h校正取食量分别为12.8, 31.8, 38.0和60.0 mg。用甘薯、白菜、大豆和南瓜叶片驯化的幼虫对初始驯化食物的选择率显著高于对玉米和麦秸的选择率;初始取食紫苏叶片的幼虫对紫苏和麦秸的选择率显著高于对玉米的选择性,初始取食玉米叶片、麦秸的幼虫分别对玉米、麦秸的选择性更高。【结论】干燥是二点委夜蛾幼虫增加对玉米摄入量的一个主要因素;二点委夜蛾适应短时高、低温的能力较强,在一定的温度范围(15-33℃),短时间内的取食量随温度升高而增加。作为干燥的一个辅助因素,温度通过影响二点委夜蛾幼虫的取食量而决定其为害玉米苗的程度;二点委夜蛾倾向于选择初孵幼虫最先接触的食物;二点委夜蛾在玉米苗上的发生为害在很大程度上取决于其自身食性。     

The Effectiveness of the Protection of Embryos and Larvae within Egg Masses of the Gastropod Epheria turritaagainst Changes in Salinity and Desiccation

The effectiveness of the protection of embryos and larvae in egg masses from reduced salinity and desiccation was investigated in the gastropod Epheria turrita, which inhabits the intertidal and upper subtidal zones. Embryos and larvae developing inside egg masses were shown not to be protected against changes in environmental salinity. Viable larvae hatched from egg masses at a salinity of 24–26. However, if free-swimming veliger larvae, rather than egg masses, were transferred to water of reduced salinity, the range of salinity tolerated by the larvae was wider, and its lower limit was 18–20. Egg masses did not protect against desiccation either. A 3-h exposure of egg masses to drying resulted in larval mortality. Larvae hatched from egg masses did not rise to the surface and displayed an early tendency towards gregarious behavior, which apparently enhances their ability to settle on a proper substrate near parental populations.     

Critical thermal limits, temperature tolerance and water balance of a sub-Antarctic kelp fly, Paractora dreuxi (Diptera: Helcomyzidae)

Paractora dreuxi displays distinct ontogenetic differences in thermal tolerance and water balance. Larvae are moderately freeze tolerant. Mean larval onset of chill coma was -5.1 degrees C, and onset of heat stupor was 35.5 degrees C. Larval supercooling point (SCP) was -3.3 degrees C with 100% recovery, although mortality was high below -4 degrees C. Starvation caused SCP depression in the larvae. Adults were significantly less tolerant, with critical thermal limits of -2.7 and 30.2 degrees C, no survival below the SCP (-9.6 degrees C), and no change in SCP with starvation. Moderate freeze tolerance in the larvae supports the contention that this strategy is common in insects from southern, oceanic islands. Fly larvae survived desiccation in dry air for 30 h, and are thus less desiccation tolerant than most other sub-Antarctic insect larvae. Water loss rates of the adults were significantly lower than those of the larvae. Lipid metabolism did not contribute significantly to water replacement in larvae, which replaced lost body water by drinking fresh water, but not sea water. Kelp fly larvae had excellent haemolymph osmoregulatory abilities. Current climate change has led to increased temperatures and decreased rainfall on Marion Island. These changes are likely to have significant effects on P. dreuxi, and pronounced physiological regulation in larvae suggests that they will be most susceptible to such change.     

Adaptive phenotypic plasticity and genetics of larval life histories in two Rana temporaria populations

Phenotypic plasticity provides means for adapting to environmental unpredictability. In terms of accelerated development in the face of pond-drying risk, phenotypic plasticity has been demonstrated in many amphibian species, but two issues of evolutionary interest remain unexplored. First, the heritable basis of plastic responses is poorly established. Second, it is not known whether interpopulational differences in capacity to respond to pond-drying risk exist, although such differences, when matched with differences in desiccation risk would provide strong evidence for local adaptation. We investigated sources of within- and among-population variation in plastic responses to simulated pond-drying risk (three desiccation treatments) in two Rana temporaria populations originating from contrasting environments: (1) high desiccation risk with weak seasonal time constraint (southern population); and (2) low desiccation risk with severe seasonal time constraint (northern population). The larvae originating from the environment with high desiccation risk responded adaptively to the fast decreasing water treatment by accelerating their development and metamorphosing earlier, but this was not the case in the larvae originating from the environment with low desiccation risk. In both populations, metamorphic size was smaller in the high-desiccation-risk treatment, but the effect was larger in the southern population. Significant additive genetic variation in development rate was found in the northern and was nearly significant in the southern population, but there was no evidence for genetic variation in plasticity for development rates in either of the populations. No genetic variation for plasticity was found either in size at metamorphosis or growth rate. All metamorphic traits were heritable, and additive genetic variances were generally somewhat higher in the southern population, although significantly so in only one trait. Dominance variances were also significant in three of four traits, but the populations did not differ. Maternal effects in metamorphic traits were generally weak in both populations. Within-environment phenotypic correlations between larval period and metamorphic size were positive and genetic correlations negative in both populations. These results suggest that adaptive phenotypic plasticity is not a species-specific fixed trait, but evolution of interpopulational differences in plastic responses are possible, although heritability of plasticity appears to be low. The lack of adaptive response to desiccation risk in northern larvae is consistent with the interpretation that selection imposed by shorter growing season has favored rapid development in north (approximately 8% faster development in north as compared to south) or a minimum metamorphic size at the expense of phenotypic plasticity.     

Survival of sheep and goat first stage protostrongylid larvae in experimental conditions: influence of humidity and temperature

The survival of first-stage larvae of a laboratory strain of Muellerius capillaris and of a natural multispecific infection (Neostrongylus linearis, Cystocaulus nigrescens, Protostrongylus rufescens) was studied for 10 to 12 day periods. The survival was estimated either on larvae in faeces or kept in tap water. Temperature (-18 degrees C to 37 degrees C) and desiccation were the ecological factors investigated. M. capillaris was the most tolerant to these factors but showed better survival at 4 degrees C (and at -18 degrees C on one occasion). N. linearis survived better at 25 degrees C or -18 degrees C and C. nigrescens at 4 degrees C and -18 degrees C. Humidification of faeces was unfavourable to the latter species. All the species could stand desiccation of faeces up to 67% of dry-matter for M. capillaris or 82% for other species. Larval survival estimated for L1 in tap water was different from that estimated for larvae in faeces. The variation in resistance to unfavourable temperatures or moisture conditions may account partly for the geographical distribution of the species.     

Survival of caddis larvae in relation to their case material in a group of temporary and permanent pools

1. The occurrence of larvae of Limnephilus coenosus and L. vittatus (Trichoptera) was recorded in 103 rock pools both before and after the dry periods in spring–summer (April–May and July–early August), when a great number of the pools dried out. In early spring, 84% of the pools sampled contained larvae of at least one of the species. Limnephilus coenosus larvae were present in a higher proportion of pools that later dried out than in the permanent ones, while L. vittatus larvae were predominantly found in permanent pools. Larvae of both species were found together in 31% of the inhabited pools.
2. Laboratory experiments, designed to elucidate differential survival under drought conditions, demonstrated that the organic cases of L. coenosus larvae held water more efficiently than did the mineral L. vittatus cases. Full-grown (fifth instar) L. coenosus larvae also survived dry conditions better than did L. vittatus larvae. The higher survival of L. coenosus was due to a combination of drought-resistant case material and a higher survival ability of the larvae themselves.
3. Limnephilus vittatus larvae were restricted by their low capability to withstand desiccation, although case material and food were available for L. vittatus larvae in all pools. Absence of case material and high-quality food in many pools, however, restricted the presence of L. coenosus larvae, but this species did not suffer from heavy mortality because of desiccation and all pool populations of L. coenosus survived until pupation.
4. The differences in species composition in these temporary and permanent pools can be explained by the differential site selection by ovipositing females, as well as by larval survival. The intrinsically greater survival of L. coenosus larvae during drought, together with the water-retaining properties of the cases, allowed this species to exploit stressful and risky habitats, such as temporary pools.