Multiple blood feeding in mosquitoes shortens the Plasmodium falciparum incubation period and increases malaria transmission potential

Many mosquito species, including the major malaria vector Anopheles gambiae, naturally undergo multiple reproductive cycles of blood feeding, egg development and egg laying in their lifespan. Such complex mosquito behavior is regularly overlooked when mosquitoes are experimentally infected with malaria parasites, limiting our ability to accurately describe potential effects on transmission. Here, we examine how Plasmodium falciparum development and transmission potential is impacted when infected mosquitoes feed an additional time. We measured P. falciparum oocyst size and performed sporozoite time course analyses to determine the parasite’s extrinsic incubation period (EIP), i.e. the time required by parasites to reach infectious sporozoite stages, in An. gambiae females blood fed either once or twice. An additional blood feed at 3 days post infection drastically accelerates oocyst growth rates, causing earlier sporozoite accumulation in the salivary glands, thereby shortening the EIP (reduction of 2.3 ± 0.4 days). Moreover, parasite growth is further accelerated in transgenic mosquitoes with reduced reproductive capacity, which mimic genetic modifications currently proposed in population suppression gene drives. We incorporate our shortened EIP values into a measure of transmission potential, the basic reproduction number R₀, and find the average R₀ is higher (range: 10.1%–12.1% increase) across sub-Saharan Africa than when using traditional EIP measurements. These data suggest that malaria elimination may be substantially more challenging and that younger mosquitoes or those with reduced reproductive ability may provide a larger contribution to infection than currently believed. Our findings have profound implications for current and future mosquito control interventions.     

The Incubation Periods of Dengue Viruses

Dengue viruses are major contributors to illness and death globally. Here we analyze the extrinsic and intrinsic incubation periods (EIP and IIP), in the mosquito and human, respectively. We identified 146 EIP observations from 8 studies and 204 IIP observations from 35 studies. These data were fitted with censored Bayesian time-to-event models. The best-fitting temperature-dependent EIP model estimated that 95% of EIPs are between 5 and 33 days at 25°C, and 2 and 15 days at 30°C, with means of 15 and 6.5 days, respectively. The mean IIP estimate was 5.9 days, with 95% expected between days 3 and 10. Differences between serotypes were not identified for either incubation period. These incubation period models should be useful in clinical diagnosis, outbreak investigation, prevention and control efforts, and mathematical modeling of dengue virus transmission.     

Fluctuations at a Low Mean Temperature Accelerate Dengue Virus Transmission by Aedes aegypti

Background

Environmental factors such as temperature can alter mosquito vector competence for arboviruses. Results from recent studies indicate that daily fluctuations around an intermediate mean temperature (26°C) reduce vector competence of Aedes aeygpti for dengue viruses (DENV). Theoretical predictions suggest that the mean temperature in combination with the magnitude of the diurnal temperature range (DTR) mediate the direction of these effects.

Methodology/Principal Findings

We tested the effect of temperature fluctuations on Ae. aegypti vector competence for DENV serotype-1 at high and low mean temperatures, and confirmed this theoretical prediction. A small DTR had no effect on vector competence around a high (30°C) mean, but a large DTR at low temperature (20°C) increased the proportion of infected mosquitoes with a disseminated infection by 60% at 21 and 28 days post-exposure compared to a constant 20°C. This effect resulted from a marked shortening of DENV extrinsic incubation period (EIP) in its mosquito vector; i.e., a decrease from 29.6 to 18.9 days under the fluctuating vs. constant temperature treatment.

Conclusions

Our results indicate that Ae. aegypti exposed to large fluctuations at low temperatures have a significantly shorter virus EIP than under constant temperature conditions at the same mean, leading to a considerably greater potential for DENV transmission. These results emphasize the value of accounting for daily temperature variation in an effort to more accurately understand and predict the risk of mosquito-borne pathogen transmission, provide a mechanism for sustained DENV transmission in endemic areas during cooler times of the year, and indicate that DENV transmission could be more efficient in temperate regions than previously anticipated.     

Association of Climatic Variability,Vector Population and Malarial Disease in District of Visakhapatnam,India: A Modeling and Prediction Analysis

Background

Malarial incidence, severity, dynamics and distribution of malaria are strongly determined by climatic factors, i.e., temperature, precipitation, and relative humidity. The objectives of the current study were to analyse and model the relationships among climate, vector and malaria disease in district of Visakhapatnam, India to understand malaria transmission mechanism (MTM).

Methodology

Epidemiological, vector and climate data were analysed for the years 2005 to 2011 in Visakhapatnam to understand the magnitude, trends and seasonal patterns of the malarial disease. Statistical software MINITAB ver. 14 was used for performing correlation, linear and multiple regression analysis.

Results/Findings

Perennial malaria disease incidence and mosquito population was observed in the district of Visakhapatnam with peaks in seasons. All the climatic variables have a significant influence on disease incidence as well as on mosquito populations. Correlation coefficient analysis, seasonal index and seasonal analysis demonstrated significant relationships among climatic factors, mosquito population and malaria disease incidence in the district of Visakhapatnam, India. Multiple regression and ARIMA (I) models are best suited models for modeling and prediction of disease incidences and mosquito population. Predicted values of average temperature, mosquito population and malarial cases increased along with the year. Developed MTM algorithm observed a major MTM cycle following the June to August rains and occurring between June to September and minor MTM cycles following March to April rains and occurring between March to April in the district of Visakhapatnam. Fluctuations in climatic factors favored an increase in mosquito populations and thereby increasing the number of malarial cases. Rainfall, temperatures (20°C to 33°C) and humidity (66% to 81%) maintained a warmer, wetter climate for mosquito growth, parasite development and malaria transmission.

Conclusions/Significance

Changes in climatic factors influence malaria directly by modifying the behaviour and geographical distribution of vectors and by changing the length of the life cycle of the parasite.     

The parasite invasion marker SRPN6 reduces sporozoite numbers in salivary glands of Anopheles gambiae

For malaria transmission to occur, Plasmodium sporozoites must infect the salivary glands of their mosquito vectors. This study reports that Anopheles gambiae SRPN6 participates in a local salivary gland epithelial response against the rodent malaria parasite, Plasmodium berghei . We showed previously that SRPN6, an immune inducible midgut invasion marker, influences ookinete development. Here we report that SRPN6 is also specifically induced in salivary glands with the onset of sporozoite invasion. The protein is located in the basal region of epithelial cells in proximity to invading sporozoites. Knockdown of SRPN6 during the late phase of sporogony by RNAi has no effect on oocyst rupture but significantly increases the number of sporozoites present in salivary glands. Despite several differences between the passage of Plasmodium through the midgut and the salivary glands, this study identifies a striking overlap in the molecular responses of these two epithelia to parasite invasion.     

Malaria's Missing Number: Calculating the Human Component of R0 by a Within-Host Mechanistic Model of Plasmodium falciparum Infection and Transmission

Human infection by malarial parasites of the genus Plasmodium begins with the bite of an infected Anopheles mosquito. Current estimates place malaria mortality at over 650,000 individuals each year, mostly in African children. Efforts to reduce disease burden can benefit from the development of mathematical models of disease transmission. To date, however, comprehensive modeling of the parameters defining human infectivity to mosquitoes has remained elusive. Here, we describe a mechanistic within-host model of Plasmodium falciparum infection in humans and pathogen transmission to the mosquito vector. Our model incorporates the entire parasite lifecycle, including the intra-erythrocytic asexual forms responsible for disease, the onset of symptoms, the development and maturation of intra-erythrocytic gametocytes that are transmissible to Anopheles mosquitoes, and human-to-mosquito infectivity. These model components were parameterized from malaria therapy data and other studies to simulate individual infections, and the ensemble of outputs was found to reproduce the full range of patient responses to infection. Using this model, we assessed human infectivity over the course of untreated infections and examined the effects in relation to transmission intensity, expressed by the basic reproduction number R₀ (defined as the number of secondary cases produced by a single typical infection in a completely susceptible population). Our studies predict that net human-to-mosquito infectivity from a single non-immune individual is on average equal to 32 fully infectious days. This estimate of mean infectivity is equivalent to calculating the human component of malarial R₀. We also predict that mean daily infectivity exceeds five percent for approximately 138 days. The mechanistic framework described herein, made available as stand-alone software, will enable investigators to conduct detailed studies into theories of malaria control, including the effects of drug treatment and drug resistance on transmission.     

Population dynamics of sporogony for Plasmodium vivax parasites from western Thailand developing within three species of colonized Anopheles mosquitoes

Background

The population dynamics of Plasmodium sporogony within mosquitoes consists of an early phase where parasite abundance decreases during the transition from gametocyte to oocyst, an intermediate phase where parasite abundance remains static as oocysts, and a later phase where parasite abundance increases during the release of progeny sporozoites from oocysts. Sporogonic development is complete when sporozoites invade the mosquito salivary glands. The dynamics and efficiency of this developmental sequence were determined in laboratory strains of Anopheles dirus, Anopheles minimus and Anopheles sawadwongporni mosquitoes for Plasmodium vivax parasites circulating naturally in western Thailand.

Methods

Mosquitoes were fed blood from 20 symptomatic Thai adults via membrane feeders. Absolute densities were estimated for macrogametocytes, round stages (= female gametes/zygotes), ookinetes, oocysts, haemolymph sporozoites and salivary gland sporozoites. From these census data, five aspects of population dynamics were analysed; 1) changes in life-stage prevalence during early sporogony, 2) kinetics of life-stage formation, 3) efficiency of life-stage transitions, 4) density relationships between successive life-stages, and 5) parasite aggregation patterns.

Results

There was no difference among the three mosquito species tested in total losses incurred by P. vivax populations during early sporogony. Averaged across all infections, parasite populations incurred a 68-fold loss in abundance, with losses of ca. 19-fold, 2-fold and 2-fold at the first (= gametogenesis/fertilization), second (= round stage transformation), and third (= ookinete migration) life-stage transitions, respectively. However, total losses varied widely among infections, ranging from 6-fold to over 2,000-fold loss. Losses during gametogenesis/fertilization accounted for most of this variability, indicating that gametocytes originating from some volunteers were more fertile than those from other volunteers. Although reasons for such variability were not determined, gametocyte fertility was not correlated with blood haematocrit, asexual parasitaemia, gametocyte density or gametocyte sex ratio. Round stages and ookinetes were present in mosquito midguts for up to 48 hours and development was asynchronous. Parasite losses during fertilization and round stage differentiation were more influenced by factors intrinsic to the parasite and/or factors in the blood, whereas ookinete losses were more strongly influenced by mosquito factors. Oocysts released sporozoites on days 12 to 14, but even by day 22 many oocysts were still present on the midgut. The per capita production was estimated to be approximately 500 sporozoites per oocyst and approximately 75% of the sporozoites released into the haemocoel successfully invaded the salivary glands.

Conclusion

The major developmental bottleneck in early sporogony occurred during the transition from macrogametocyte to round stage. Sporozoite invasion into the salivary glands was very efficient. Information on the natural population dynamics of sporogony within malaria-endemic areas may benefit intervention strategies that target early sporogony (e.g., transmission blocking vaccines, transgenic mosquitoes).     

A reaction–diffusion malaria model with incubation period in the vector population

Malaria is one of the most important parasitic infections in humans and more than two billion people are at risk every year. To understand how the spatial heterogeneity and extrinsic incubation period (EIP) of the parasite within the mosquito affect the dynamics of malaria epidemiology, we propose a nonlocal and time-delayed reaction–diffusion model. We then define the basic reproduction ratio R₀{\mathcal{R}_0} and show that R₀{\mathcal{R}_0} serves as a threshold parameter that predicts whether malaria will spread. Furthermore, a sufficient condition is obtained to guarantee that the disease will stabilize at a positive steady state eventually in the case where all the parameters are spatially independent. Numerically, we show that the use of the spatially averaged system may highly underestimate the malaria risk. The spatially heterogeneous framework in this paper can be used to design the spatial allocation of control resources.     

Modeling Within-Host Effects of Drugs on Plasmodium falciparum Transmission and Prospects for Malaria Elimination

Achieving a theoretical foundation for malaria elimination will require a detailed understanding of the quantitative relationships between patient treatment-seeking behavior, treatment coverage, and the effects of curative therapies that also block Plasmodium parasite transmission to mosquito vectors. Here, we report a mechanistic, within-host mathematical model that uses pharmacokinetic (PK) and pharmacodynamic (PD) data to simulate the effects of artemisinin-based combination therapies (ACTs) on Plasmodium falciparum transmission. To contextualize this model, we created a set of global maps of the fold reductions that would be necessary to reduce the malaria R_C (i.e. its basic reproductive number under control) to below 1 and thus interrupt transmission. This modeling was applied to low-transmission settings, defined as having a R₀<10 based on 2010 data. Our modeling predicts that treating 93–98% of symptomatic infections with an ACT within five days of fever onset would interrupt malaria transmission for ∼91% of the at-risk population of Southeast Asia and ∼74% of the global at-risk population, and lead these populations towards malaria elimination. This level of treatment coverage corresponds to an estimated 81–85% of all infected individuals in these settings. At this coverage level with ACTs, the addition of the gametocytocidal agent primaquine affords no major gains in transmission reduction. Indeed, we estimate that it would require switching ∼180 people from ACTs to ACTs plus primaquine to achieve the same transmission reduction as switching a single individual from untreated to treated with ACTs. Our model thus predicts that the addition of gametocytocidal drugs to treatment regimens provides very small population-wide benefits and that the focus of control efforts in Southeast Asia should be on increasing prompt ACT coverage. Prospects for elimination in much of Sub-Saharan Africa appear far less favorable currently, due to high rates of infection and less frequent and less rapid treatment.     

Rhoptry neck protein 11 has crucial roles during malaria parasite sporozoite invasion of salivary glands and hepatocytes

The malaria parasite sporozoite sequentially invades mosquito salivary glands and mammalian hepatocytes; and is the Plasmodium lifecycle infective form mediating parasite transmission by the mosquito vector. The identification of several sporozoite-specific secretory proteins involved in invasion has revealed that sporozoite motility and specific recognition of target cells are crucial for transmission. It has also been demonstrated that some components of the invasion machinery are conserved between erythrocytic asexual and transmission stage parasites. The application of a sporozoite stage-specific gene knockdown system in the rodent malaria parasite, Plasmodium berghei, enables us to investigate the roles of such proteins previously intractable to study due to their essentiality for asexual intraerythrocytic stage development, the stage at which transgenic parasites are derived. Here, we focused on the rhoptry neck protein 11 (RON11) that contains multiple transmembrane domains and putative calcium-binding EF-hand domains. PbRON11 is localised to rhoptry organelles in both merozoites and sporozoites. To repress PbRON11 expression exclusively in sporozoites, we produced transgenic parasites using a promoter-swapping strategy. PbRON11-repressed sporozoites showed significant reduction in attachment and motility in vitro, and consequently failed to efficiently invade salivary glands. PbRON11 was also determined to be essential for sporozoite infection of the liver, the first step during transmission to the vertebrate host. RON11 is demonstrated to be crucial for sporozoite invasion of both target host cells – mosquito salivary glands and mammalian hepatocytes – via involvement in sporozoite motility.     

Annotated Differentially Expressed Salivary Proteins of Susceptible and Insecticide-Resistant Mosquitoes of Anopheles stephensi

Vector control is one of the major global strategies for control of malaria. However, the major obstacle for vector control is the development of multiple resistances to organochlorine, organophosphorus insecticides and pyrethroids that are currently being used in public health for spraying and in bednets. Salivary glands of vectors are the first target organ for human-vector contact during biting and parasite-vector contact prior to parasite development in the mosquito midguts. The salivary glands secrete anti-haemostatic, anti-inflammatory biologically active molecules to facilitate blood feeding from the host and also inadvertently inject malaria parasites into the vertebrate host. The Anopheles stephensi mosquito, an urban vector of malaria to both human and rodent species has been identified as a reference laboratory model to study mosquito—parasite interactions. In this study, we adopted a conventional proteomic approach of 2D-electrophoresis coupled with MALDI-TOF mass spectrometry and bioinformatics to identify putative differentially expressed annotated functional salivary proteins between An. stephensi susceptible and multiresistant strains with same genetic background. Our results show 2D gel profile and MALDI-TOF comparisons that identified 31 differentially expressed putative modulated proteins in deltamethrin/DDT resistant strains of An. stephensi. Among these 15 proteins were found to be upregulated and 16 proteins were downregulated. Our studies interpret that An. stephensi (multiresistant) caused an upregulated expression of proteins and enzymes like cytochrome 450, short chain dehyrdogenase reductase, phosphodiesterase etc that may have an impact in insecticide resistance and xenobiotic detoxification. Our study elucidates a proteomic response of salivary glands differentially regulated proteins in response to insecticide resistance development which include structural, redox and regulatory enzymes of several pathways. These identified proteins may play a role in regulating mosquito biting behavior patterns and may have implications in the development of malaria parasites in resistant mosquitoes during parasite transmission.     

The Plasmodium vivax Merozoite Surface Protein 3β Sequence Reveals Contrasting Parasite Populations in Southern and Northwestern Thailand

Background

Malaria control efforts have a significant impact on the epidemiology and parasite population dynamics. In countries aiming for malaria elimination, malaria transmission may be restricted to limited transmission hot spots, where parasite populations may be isolated from each other and experience different selection forces. Here we aim to examine the Plasmodium vivax population divergence in geographically isolated transmission zones in Thailand.

Methodology

We employed the P. vivax merozoite surface protein 3β (PvMSP3β) as a molecular marker for characterizing P. vivax populations based on the extensive diversity of this gene in Southeast Asian parasite populations. To examine two parasite populations with different transmission levels in Thailand, we obtained 45 P. vivax isolates from Tak Province, northwestern Thailand, where the annual parasite incidence (API) was more than 2%, and 28 isolates from Yala and Narathiwat Provinces, southern Thailand, where the API was less than 0.02%. We sequenced the PvMSP3β gene and examined its genetic diversity and molecular evolution between the parasite populations.

Principal Findings

Of 58 isolates containing single PvMSP3β alleles, 31 sequence types were identified. The overall haplotype diversity was 0.77±0.06 and nucleotide diversity 0.0877±0.0054. The northwestern vivax malaria population exhibited extensive haplotype diversity (HD) of PvMSP3β (HD = 1.0). In contrast, the southern parasite population displayed a single PvMSP3β allele (HD = 0), suggesting a clonal population expansion. This result revealed that the extent of allelic diversity in P. vivax populations in Thailand varies among endemic areas.

Conclusion

Malaria parasite populations in a given region may vary significantly in genetic diversity, which may be the result of control and influenced by the magnitude of malaria transmission intensity. This is an issue that should be taken into account for the implementation of P. vivax control measures such as drug policy and vaccine development.     

Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.     

Persistence of Viruses in Desert Soils Amended with Anaerobically Digested Sewage Sludge

Pima County, Ariz., is currently investigating the potential benefits of land application of sewage sludge. To assess risks associated with the presence of pathogenic enteric viruses present in the sludge, laboratory studies were conducted to measure the inactivation rate (k = log₁₀ reduction per day) of poliovirus type 1 and bacteriophages MS2 and PRD-1 in two sludge-amended desert agricultural soils (Brazito Sandy Loam and Pima Clay Loam). Under constant moisture (approximately -0.05 × 10⁵ Pa for both soils) and temperatures of 15, 27, and 40°C, the main factors controlling the inactivation of these viruses were soil temperature and texture. As the temperature increased from 15 to 40°C, the inactivation rate increased significantly for poliovirus and MS2, whereas, for PRD-1, a significant increase in the inactivation rate was observed only at 40°C. Clay loam soils afforded more protection to all three viruses than sandy soils. At 15°C, the inactivation rate for MS2 ranged from 0.366 to 0.394 log₁₀ reduction per day in clay loam and sandy loam soils, respectively. At 27°C, this rate increased to 0.629 log₁₀ reduction per day in clay loam soil and to 0.652 in sandy loam soil. A similar trend was observed for poliovirus at 15°C (k = 0.064 log₁₀ reduction per day, clay loam; k = 0.095 log₁₀ reduction per day, sandy loam) and 27°C (k = 0.133 log₁₀ reduction per day, clay loam; k = 0.154 log₁₀ reduction per day, sandy loam). Neither MS2 nor poliovirus was recovered after 24 h at 40°C. No reduction of PRD-1 was observed after 28 days at 15°C and after 16 days at 27°C. At 40°C, the inactivation rates were 0.208 log₁₀ reduction per day in amended clay loam soil and 0.282 log₁₀ reduction per day in sandy loam soil. Evaporation to less than 5% soil moisture completely inactivated all three viruses within 7 days at 15°C, within 3 days at 27°C, and within 2 days at 40°C regardless of soil type. This suggests that a combination of high soil temperature and rapid loss of soil moisture will significantly reduce risks caused by viruses in sludge.     

Inactivation of Ascaris Eggs in Source-Separated Urine and Feces by Ammonia at Ambient Temperatures

Sustainable management of toilet waste must prevent disease transmission but allow reuse of plant nutrients. Inactivation of uterus-derived Ascaris suum eggs was studied in relation to ammonia in source-separated urine without additives and in human feces to which urea had been added, in order to evaluate ammonia-based sanitation for production of safe fertilizers from human excreta. Urine was used concentrated or diluted 1:1 and 1:3 with tap water at 4, 14, 24, and 34°C. Fecal material, with and without ash, was treated with 1% or 2% (wt/wt) urea at 24 and 34°C. At 34°C eggs were inactivated in less than 10 days in urine and in amended feces. At 24°C only feces with 2% (wt/wt) urea or 1% (wt/wt) urea at high pH (10) inactivated all eggs within 1 month, and no inactivation was observed after 75 days in urine diluted 1:3 (18 ± 11 mM NH₃). At temperatures of ≥24°C, NH₃ proved to be an efficient sanitizing agent in urine and feces at concentrations of ≥60 mM. Treating fecal material at 34°C can give a 6-log₁₀ egg inactivation within 1 month, whereas at 24°C 6 months of treatment is necessary for the same level of egg inactivation. At temperatures of 14°C and below, inactivation rates were low, with viable eggs after 6 months even in concentrated urine.     

Potential effects of warmer worms and vectors on onchocerciasis transmission in West Africa

Development times of eggs, larvae and pupae of vectors of onchocerciasis (Simulium spp.) and of Onchocerca volvulus larvae within the adult females of the vectors decrease with increasing temperature. At and above 25°C, the parasite could reach its infective stage in less than 7 days when vectors could transmit after only two gonotrophic cycles. After incorporating exponential functions for vector development into a novel blackfly population model, it was predicted that fly numbers in Liberia and Ghana would peak at air temperatures of 29°C and 34°C, about 3°C and 7°C above current monthly averages, respectively; parous rates of forest flies (Liberia) would peak at 29°C and of savannah flies (Ghana) at 30°C. Small temperature increases (less than 2°C) might lead to changes in geographical distributions of different vector taxa. When the new model was linked to an existing framework for the population dynamics of onchocerciasis in humans and vectors, transmission rates and worm loads were projected to increase with temperature to at least 33°C. By contrast, analyses of field data on forest flies in Liberia and savannah flies in Ghana, in relation to regional climate change predictions, suggested, on the basis of simple regressions, that 13–41% decreases in fly numbers would be expected between the present and before 2040. Further research is needed to reconcile these conflicting conclusions.     

Effect of growth temperature and temperature shifts on spinach leaf morphology and photosynthesis

The growth kinetics of spinach plants (Spinacia oleracea L. cv Savoy) grown at 5°C or 16°C were determined to allow us to compare leaf tissues of the same developmental stage rather than chronological age. The second leaf pairs reached full expansion at a plant age of 32 and 92 days for the 16°C and 5°C plants, respectively. Growth at 5°C resulted in an increased leaf area, dry weight, dry weight per area, and leaf thickness. Despite these changes, pigment content and composition, room temperature in vivo fluorescence, and apparent quantum yield and light-saturated rates of CO₂ exchange or O₂ evolution were not affected by the growth temperature. Furthermore, 5°C expanded leaves were found to be more resistant to photoinhibition at 5°C than were 16°C expanded leaves. Thus, it is concluded that spinach grown at low temperature is not stressed. However, shifting spinach leaves from 5°C to 16°C or from 16°C to 5°C for 12 days after full leaf expansion had occurred resulted in a 20 to 25% reduction in apparent quantum yields and 50 to 60% reduction in light saturated rates of both CO₂ exchange and O₂ evolution. This was not accompanied by a change in the pigment content or composition or in the room temperature in vivo fluorescence. It appears that leaf aging during the temperature shift period can account for the reduction in photosynthesis. Comparison of cold-hardened and non-hardened winter rye (Secale cereale L. cv Muskateer) with spinach by in vivo fluorescence indicated that rye is more sensitive to both short term and longer duration temperature shifts than is spinach. Thus, susceptibility to an abrupt temperature shift appears to be species dependent.     

Factors Influencing the Induction of Freezing Tolerance by Abscisic Acid in Cell Suspension Cultures of Bromus inermis Leyss and Medicago sativa L

A 2-gram fresh weight inoculum of bromegrass (Bromus inermis Leyss. culture BG970) cell suspension culture treated with 7.5 × 10⁻⁵ molar abscisic acid (ABA) for 7 days at 25°C survived slow cooling to −60°C. Over 80% of the cells in ABA treated cultures survived immersion in liquid N₂ after slow cooling to −40 or −60°C. In contrast, a 6-gram fresh weight inoculum only attained a hardiness level of −28°C after 5 days of ABA treatment. Ethanol (2 × 10⁻² molar) added to the culture medium at the time of ABA addition, inhibited the freezing tolerance of bromegrass cells by 25°C. A 6-gram inoculum of both control and ABA treated bromegrass cells altered the pH of the medium more than a 2-gram inoculum. ABA inhibited the increase in fresh weight of bromegrass by 20% after 4 days. Both control and ABA (10⁻⁴ molar) treated alfalfa cells (Medicago sativa L.) grown at 25°C hardened from an initial LT₅₀ of −5°C to an LT₅₀ of −23°C by the third to fifth day after subculture. Thereafter, the cells dehardened but the ABA treated cells did not deharden to the same level as the control cells. ABA inhibited the increase in fresh weight of alfalfa by 50% after 5 days.