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How It Controls Fatty Acid Profiles Isotope Signatures And Stoichiometry In The Malaria Mosquito Anopheles Arabiensis An Analysis Of Diet Quality

deionised distilled waterKnowing the underlying mechanisms of mosquito ecology will ensure effective vector management and contribute to the overall goal of malaria control.

Mosquito populations show a high degree of population plasticity in response to environmental variability. Eventually, the principle factors controlling population size and fecundity are for the most part unknown. Larval habitat and diet play a crucial role in subsequent mosquito fitness. Information gained from this process offers us some insight into the mechanisms and processes taking place in natural native mosquito habitats, Developing the most competitive insects for sterile insect technique programmes requires a production orientated perspective, to deduce the most effective larval diet formulation.

Fatty acid profiles and de novo or direct assimilation pathways, of wholeindividual mosquitoes reared on a range of larval diets were determined using pyrolysis gas chromatograph/mass spectrometry.

We used elemental analysis and isotope ratio mass spectrometry to measure individualwholebody carbon, nitrogen and phosphorous values and to assess the impact of dietary quality on subsequent population stoichiometry, size, quality and isotopic signature. Diet had the greatest impact on fatty acid profiles of the mosquitoes, that exhibited a high degree of dietary routing, characteristic of generalist feeders. De novo synthesis of loads of important FAs was observed. You should take it into account. Mosquito N stoichiometry was fixed in the teneral stage. Dietary N content had significant influence on mosquito size, and P was shown to be a flexible pool which limited overall population size.

Direct routing of FAs was evident but there was ubiquitous denovo synthesis suggesting mosquito larvae are competent generalist feeders capable of survival on diet with varying characteristics.

It was concluded that nitrogen availability in the larval diet controlled teneral mosquito size and that teneral CN ratio is a ‘sex and’ ‘species specific’ fixed parameter. This finding has significant implications for overall mosquito competitiveness and environmental management. Understanding mosquito ecology has recently been prioritized as a prerequisite for malaria eradication. Ferguson et al.

Prey and competitors, an understanding of their ecology is essential to avoid any interventions triggering cascades of ecological effects that could lead to enhanced malaria transmission, as mosquito vectors are embedded within ecological communities as predators.

Understanding of the competitive interactions and species specific niche adaptations is critical for effective vector management, with over thirty different primary vectors dominating transmission. Resulting in smaller, more robust and fecund populations, the mechanisms underlying this plasticity are largely unexplored,, even if many studies have shown that the growth rates of larval mosquito vectors are negatively correlated with their population size. Body size has also been shown to have important fitness implications, however individual body size frequency distributions within a population remain underinvestigated in insects mostly. Then, the key factors controlling population dynamics and body size is larval nutrition, and previous studies have shown that nitrogen and phosphorus availabilities are important ecological determinants in other insects,. Usually, it’s extremely difficult to study nutritional impacts on such small insects and generally methods of analysis are laborious and complex, often limiting the scope of the studies conducted. Here we present some rapid techniques that may overcome some amount of these constraints opening up opportunities for more holistic ecosystem based research.

Advances in elemental analysis and pyrolysis techniques to measure fatty acid concentrations, mean that it’s now possible to investigate nutritional impacts on mosquito larvae development and survival on an individual basis.

This allows us to explore mosquito larval development within the larger ecological framework and relate it to current paradigms in ecological thinking, similar to ecological stoichiometry. Ecological stoichiometry is heralded as the unifying theory of ecology. It is on the basis of simple laws of physics like mass balance and energy dissipation meshed with the biological fundamentals of energy tradeoffs at biochemical and individual levels. Just think for a moment. These basics have been cleverly honed to explain the dynamics of individuals, populations, communities and ecosystems.

At the very base of ecological stoichiometry theory is the concept that at the organism level there’s an unique balance of multiple chemical substances, mainly ratios of carbon.

Usually not exclusively, nitrogen or phosphorous,, Fundamentally the theory suggests that living organisms are constrained and different from their environment, and in almost all circumstances going to be limited by one element. Little is known of the extent to which stoichiometry drives population dynamics and its consequences for general mosquito biology, albeit this is an universal phenomenon. Stoichiometric theory contrasts to the current theory that mosquito larval nutrition is a complex combination of dietary requirements. In this study we set out to test whether these theories hold up for Anopheles arabiensis mosquitoes and whether they might explain some observed phenomenon of population plasticity.

Undoubtedly it’s known that proteins, sugar, polyunsaturated fatty acids, sterols, vitamins and nucleotides are all essential for mosquito development, Mosquito larval nutrition is extensively studied.

It had been shown that at least fourteen amino acids are essential for larval growth and survival,,. Additionally, minimal concentrations of essential vitamins are required to ensure optimal growth of a few mosquito species,. Notice that dadd Kleinjan also showed that less that 5percent of Cx. It is well documented that mosquito larval diet quality and quantity influences both adult quality and in turn sexual competitiveness,. Efficient and economic mass rearing virtually that systems are generally constrained by specific macronutritional requirements which have the individual components embedded within them, and that primary producers and thus secondary and higher level consumers are ultimately constrained by biogeochemistry. It is well documented that primary producers respond positively to inputs of nitrogen and phosphorous, as we are looking at the limiting elements in most natural systems. Extending from this, evolutionary theory states that the fittest individuals will use the available resources for reproduction most efficiently and therefore their genes will dominate. Actually, evolutionary logic should suggest that generalist secondary consumers will thus adapt to utilize the most commonly present components in primary producers and that these would’ve been used in the most energy efficient manner,. Indeed current trophic interaction research suggests that it’s energetically more efficient to incorporate dietary fatty acids directly into the consumer’s tissue without degradation or modification, a process termed dietary routing.

Fatty acid profiles provide a lots of information on the development, reproduction, health and feeding ecology of organisms,. Previous studies have stressed that mosquitoes are unable to elongate the 18C poly unsaturated FAs and thus C18, C20 and C22 polyunsaturated fatty acids are essential for larval development, adult survival and flight. In natural aquatic environments these fatty acids can be present and are possibly not limiting constraints of larval nutrition per se. Did you hear of something like this before, this is the case right? In most ecological systems energy transfer and energy flux is thought to be the primary constraint on total ecosystem productivity. Therefore, here we present a framework with which we test the hypothesis that dietary quality, or nutrient content, stoichiometry, plays a significant role in regulating ecosystem energy and nutrient transfers in primary consumers and can be used as a predictive tool of population response.

Studying larval nutrition within the context of developing diets for mass reared anopheles mosquitoes gave us the opportunity to test heaps of hypotheses that should support or discredit the stoichiometry theory. The pyrolysis GCMS system offered us the chance to engage in pretty simple comprehensive analysis of the fatty acids in the diet and their effects on individual mosquito FA profiles. Whenever giving us insight into the important features of diet composition, Elemental analysis and isotope ratio mass spectrometry of whole mosquitoes meant that we could compare whole body carbon, nitrogen and phosphorous and macro nutrient levels, in addition to the fatty acid profile of the diet. While allowing us to confidently apply these techniques in future field studies, the isotope analysis enabled us to investigate quite a few current assumptions in isotope ecology and test those assumptions in controlled environments. Central null hypotheses we set out to explore were.

With LD 12∶12 h photoperiod, all experiments were conducted at the Insect Pest Control Laboratory in Seibersdorf. In climate controlled rooms maintained at 27°C ±1°C and 60 RH ±10percentage, including dusk and dawn.

Anopheles arabiensis ‘MRA856’, was used in all the experiments. While having originated from Dongola, fed on identical diet. Less than 4 hours after eclosion, 32 larvae were transferred into six 9 cm diameter Petri dishes containing 32 deionised ml water and these were fed daily with 2 ml of a 1, 15, or 2percent solution of a KD1 diet which was a mixture of ground wheat, corn, bean, chick pea, rice, bovine liver powder and Vita mix in the following ratio. Six replicate dishes were set up for every treatment. Just think for a moment. All pupae were collected daily and live un fed teneral adults collected within 12 hours. Now look. Wing length was determined for every specimen. Measurements were performed with AnalySIS FIVE software, Wing length was measured from the alula notch to the wing tip. Wings were re united with their bodies in the tin cups used for analysis and thus whole adult mosquitoes might be analysed for total body carbon and nitrogen and their isotopic ratios as described below. Also, this meant TBC and TBN may be compared against wing length at the amount of individual mosquitoes.

In this experiment we set out to determine which factors of nutritional quality could influence mosquito size and whether fatty acids were directly routed from diet to the consuming larva and consequently preserved in the adult mosquito. The sixteen diets tested in this experiments were AP100, bean powder, bovine liver powder, brewer’s yeast, carrot, chick pea, corn, rice, soy hydrolysate, spirulina, squid liver powder, tuna, vitamin mix, wheat, wheat bran and yeast hydrolysate.

Materials and Methods.

Five hundred larvae were counted into a tray containing 5 L of ‘deionized’ water. Due to the sensitivities of anopheline species to overfeeding and larval habitat fouling, the mosquitoes were fed on demand. This diet was added in a ground form in quantities aiming to attain maximum adult survival on the basis of the colour and state of the larval water and the previous experience of the technicians. Newly formed pupae were transferred to emergence tubes. Furthermore, upon adult emergence, ten males and ten females were transferred to Eppendorf tubes and frozen. Although, care was taken to sample the first ten males and first ten females that emerged from every treatment to overcome any emergence date bias. These were randomly divided into three batches and triplicate whole mosquito samples of every sex were analysed for fatty acids, TBN and TBC and their respective 15N and 13C values, and TBP.

This experiment was set up to determine the influence of dietary N and P concentration on adult and pupal survival. Triplicate sets of 16 1st instar larvae were loaded into 35 mm diameter petri dishes containing 16 water ml. Each dish daily received 1 a ml 1 solution of one the sixteen different larval foods listed above. For instance, it is shown in previous experiments that a 1 concentration was the concentration where sufficient food was available but was least going to produce water fouling and associated effect on the population size. Now pay attention please. Pupation date was noted and adults collected as described above.

Typically 100 diet µg or a complete mosquito specimen were put into a quartz tube and 4 µl of a diluted, aqueous solution of tetramethylammonium hydroxide was added. The samples were subsequently pyrolyzed at 450°C for 10 s with a CDS 5250 pyrolysis autosampler attached to a Thermo Trace GC Ultra/MD 800 gas chromatography/mass spectrometry system. With a couple of unsaturated fatty acids, volatile products were separated on a Supelco SP 2330 column with helium 6 as carrier gas to 230°C at a rate of 10°C min−The mass spectrometer was operated in EI mode at a source temperature of 200° The method was optimised depending on the standard linseed oil, a triglyceride based oil. An optimal thermally assisted hydrolysis and methylation method was developed to avoid the known problems of isomerisation.

Py GCMS’ requires no sample preparation apart from the addition of TMAH.

Analysis typically takes 20 minutes per sample and 100 µg C is the ideal sample size. Notice, while following a complex procedure of lipid extraction, purification, transesterification and methylation of approximately ’50 70′ mg of sample, In contrast fatty acids are conventionally measured by gas liquid chromatography using a flame ionisation detector. Consequently, the sample preparation procedure typically takes 23 days. Notice, whenever using an isotope ratio mass spectrometer interfaced with an elemental analyzer, Whole single mosquito samples and ground diet samples were dried at 60°C for 24 h, placed into 8 by 5 mm tin cups and analyzed at SILVER, Vienna University for total N, C,15N and 13C. That is interesting. Samples were combusted in an atmosphere of oxygen at 1020°C and passed over chromium oxide and silvered cobalt oxide for complete oxidation, and subsequently over hot copper to reduce oxides of nitrogen to elemental nitrogen. There is a lot more information about this stuff here. The resultant gas was carried in a stream of helium through a scrubber to remove residual water and was hereafter passed over a gas chromatographic column to separate N2 and COPeaks were bled into the mass spectrometer to determine the isotopic ratios. The isotope ratios were expressed as parts per thousand per mille or δ deviation from the internationally recognized standards, Vienna Pee Dee Belemnite and atmospheric nitrogen. Similarly the deviation from VPDB multiplied by a thousand gives the delta notation for 13C.

The pyrolysis method gave similar patterns of relative fatty acid composition of the three diets BLP, SLP and Tuna compared to the conventional method, revealed using simple regression analysis as was previously demonstrated. On the basis of these results, it was impossible to compare measurements of single mosquitoes using the conventional methods as long as the constraints described in the methods section, previous conclusions, and the appropriate analysis of standards it was assumed that the method was suitable for the analysis of FAs in single mosquitoes. The wheat spectra were unusual in that only one fatty acid.

The most common fatty acids present in all the diets were the palmitic ), palmitoleic, stearic, oleic and linoleic acids.

Spirulina contained the characteristic gamma linolenic acid. Generally, whenever, in most cases accounting for So there’s preferential accumulation from the diet of palmitoleic and oleic FAs by the mosquitoes.

Matrix of mosquito and diet versus relative fatty acid composition was constructed, with intention to establish whether you are what you eat.

With Euclidean distances of less than At the next level the mosquitoes fed very similar diet showed the greatest degree of similarity, at the first level, mosquitoes of identical sex which were fed identical diet showed the highest degree of similarity, at the next level it appeared that there was some clustering on the basis of if the mosquitoes were fed a cereal, fish or legume diet. Whenever suggesting that there’s a high degree of nutritional plasticity and providing strong evidence for dietary routing, diet had a stronger influence than gender on the fatty acid profile of the individual mosquitoes. As a result, this led to the rejection of hypothesis 1 that fatty acid composition of mosquitoes is fixed and not influenced by the diet.

In an attempt to generate comparable information from the large data set and reveal the flow and synthesis of individual fatty acids up the food chain, graphically evident from Figure 3, in very straightforward mathematical manner, both the food and the mosquitoes were scored determined by the presence was computed.

They’ve been scored with a 1 and designated denovo synthesis, if the value for mosquitoes was greater than the value of diet. The sum of these values was computed for any fatty acid and calculated as a percentage of the overall amount of diets used in the overall analysis, so this allowed male and female values to be compared. If the sum of the binary value of the mosquitoes and the binary value of the factorised diet was greater than number of mosquitoes used in the analysis it was assumed that there was direct uptake and a value of 1 was assigned and again calculated as a percentage of the total number of diets used for both males and females, with an intention to determine the occurrence of direct uptake. As if there was no fatty acid present in the diet the factorised value of the diet my be zero, with that said, this has pretty straightforward logic and thus only when the fatty acid was present in both the diet and the mosquito should the sum of the values be greater than the total amount of mosquitoes used, and thus the number 1 can be assigned to indicate direct uptake.

Graphic showing the occurrence of ‘denovo’ synthesis or direct uptake as a percentage of the total population analysed. NB. Any number under 100 indicates the fatty acid was not present in all the Anopheles arabiensis mosquitoes analysed. We’re looking at all common fatty acids found in a range of food stuffs,, The fatty acids present in all the mosquitoes were the palmitic, palmitoleic, stearic, oleic and linoleic acids. The analysis suggested that these were all directly taken up from the diet, with that said, this fits the hypothesis of dietary routing, that suggests that organisms will take up and use FAs in their original form to avoid energy loss or the cost associated with modification.

By producing a matrix of any average diet RFAC versus average mosquito RFAC, in order to further examine the amount of dietary routing a cluster analysis was performed on the raw data of the 28 possible fatty acids. This allowed us to estimate, on an individual fatty acid basis, the apparent transfer and conservation of RFAC profile up the food chain from a range of diets. For this analysis resemblance matrices were constructed on the basis of Bray Curtis similarity. Anyway, in female mosquitoes the highest diet to mosquito similarities were in the linoleic t linoleic c, oleic palmitic and rumelenic, gamma linolenic fatty acids. Oftentimes with only the rumelenic and gamma linolenic not present in the mosquitoes and diets analysed, it might be argued that there are also the fatty acids most commonly found in both diets and the mosquitoes analysed. Certainly, in the males the highest diet to mosquito similarities were in palmitic rumelenic, gamma linolenic, argaric acids. Of course the percentage occurrence of ‘denovo’ synthesis was similar for rumelenic and gamma linolenic acids in both males and females. There is more info about it on this website. Argaric acid was exclusively produced by denovo synthesis in only 30percentage of the females and 54 of males, and obtained by direct uptake in about 5 of males from the squid liver powder treatment. Essentially, they’ve been not successfully analysed for fatty acids due to technical problems, possibly leading to this discrepancy, albeit females of the SLP treatment were viable and of average size depending on total body carbon data.

In experiment three percentage P in the diet appeared to have a greater impact on adult production per dish than dietary N, both showing weak but highly significant correlations.

While wing length, survival and possibly fecundity in An, These analyses taken together suggest that larval dietary quality and quantity have a substantial impact on adult population size. We have shown that fatty acids just like arachidonic acid which have previously been shown to be essential for Culex species were only present in around 80 of male and 60percent of female mosquitoes with denovo synthesis evident in at least 50 of the mosquitoes sampled,. While suggesting that these acids may play a role in the synthesis of arachidonic acid in mosquitoes, The highest levels of similarity between mosquito arachidonic RFAC and diet were in steric acid in the males and eicosaptaenoic acid in the females. Anyway, wheat bran had high levels of linoleic acid which is an established precursor of arachidonic acid, In addition there were high levels of ‘de novo’ synthesis of arachidonic acid in the wheat bran fed mosquitoes. Anyways, it can be found in some fungi, mosses and ferns and is a major component of a few microalgae, where it reaches up to 47percentage of the triglyceride pool,. Eventually, this could explain the link with mosquito larval nutrition, macrobenthic algae being omnipresent in most natural larval ecosystems. Previous work has suggested that mosquitoes have to obtain the C1822 fatty acids from their diet as they are unable to elongate the 18C acids,,,. Consequently, however in these experiments there was substantial evidence of ‘denovo’ synthesis in the 18C plus group, in both males and females, They have shown that eicosapentaenoic acid, docosahexaenoic acid and arachidonic acid,, are all essential acids. Whether it’s the result of elongation or shortening, by using individually stable isotope labelled fatty acids and a pyrolysis system linked to an isotope ratio mass spectrometer it may be possible to further elucidate these pathways, is unclear from our results. With the caveat that these experiments did not study the full life cycle of these An, only decohexaenoic acid in both males and females and gadoleic acid in males. Suggesting they are not essential. These fatty acids previously identified as essential for mosquitoes might be necessary to complete the full life cycle.

It appears that mosquito size and consequent competitiveness is controlled by the nitrogen content and maybe more importantly nutritional bioavailability of that nitrogen from the larval food source, when we combine the evidence from all the experiments presented.

Conversely, concentration of P is often quoted as the degree of overall productivity of the aquatic system. The general stoichiometric mismatch between diet and mosquitoes should suggest that nitrogen is indeed limiting both in the laboratory and the natural environment. While indicating that maybe these larvae were carbon rather than nitrogen limited, In the tuna meal, squid liver powder, and brewer’s yeast treatments this was not the case. It is it’s unlikely that these dietary configurations my be observed in nature. We hypothesise that in An. Let me tell you something. Larval overfeeding often leads to high larval mortality and An. Indeed additional simple chemical analysis showed that larval water ammonium concentration in the healthy arabiensis trays was around 2 ppm NH+4 compared to 100 ppm NH+4 in the larval trays of albopictus species.

Is also a more flexible storage component, evident from the data shown in Figure This hypothesis would explain why lots of us know that there is little correlation between total body C and P values but a strong correlation between fatty acid profiles and total body P, contrary to stoichiometric theory, we hypothesise that total larval nitrogen availability linearly determines the overall size of the mosquito which ranged from 20 µg N/80 µg C to 58 µg N/261 µg C. That P ain’t only present as a structural component linked to specific phospholipids.

In essence we reject as it appears that teneral N ratios are fairly fixed with values of 2∶1 and 5∶1 and percentage SD of less than 7 and 11 for males and females, hypothesis 2 and replace it with Teneral individual N is fixed and not influenced by the larval diet respectively. Also, it might be argued that stoichiometric theory is more applicable to aquatic environments but these mosquitoes were sampled as nonfed adults and as such were not subjected to a terrestrial dietary environment. Also, some explanation could’ve been offered by the terrestrial feeding ecology of the mosquito.

Or even having a flexible P store, dietary restriction. This mechanism of N determining size, and P determining longevity and mosquito abundance will result in an evolutionarily successful flexible trade off strategy between size and longevity. On top of this, small mosquitoes live longer increasing probability of finding a mate, whereas larger individuals play hard, win the mate and die young, as is shown in other insect species similar to crickets. Body size is often strongly correlated with fighting ability, or resource holding potential, such that the larger of two competing males usually wins the contest,. Considering the above said. Larger male mosquitoes been reported to be more successful in mating than smaller ones,. Intriguingly female body size has also an advantage in mate selection, larger females of An. Certainly, field studies have noted a positive correlation between female body size, that is presumably influenced by larval nutrition and competition, and parity status,.

Dietary restriction in insects has generally been shown to increase longevity, and longevity can in turn lead to an increased chance of mating. As yet unpublished, indeed this flexible hypothesis for mosquitoes is backed up by a very recent study of Anopheles gambiae in which mean adult male body size significantly influenced adult survival. However they also found that dietary P had little influence on cricket life span, Males that consumed the greatest amounts of food had the lowest survival. That said, these contradictory findings could reflect the extremely different life cycles of cricket and mosquitoes. Moreover failing to demonstrate strict stoichiometric interactions, they also found that neither total body carbon nor nitrogen were correlated with total body phosphorous, indeed what the authors did stress was that there was a significant relationship between total body carbon and nitrogen. Whenever suggesting that P stores are used up as insects get older and are not replenished, In line with our hypothesis they did find that older insects were more depleted in P. Woods et al. They suggested that a couple of taxa exhibit inverse dependence of P content on body size. Now please pay attention. Nielsen et al.

Pyrolysis GCMS allowed a comprehensive analysis of fatty acid profiles of single mosquitoes and their diets to be undertaken which revealed a regular occurrence of ‘de novo’ synthesis of a lot of important fatty acids. It also suggested that fatty acids play an important role in the P nutrition of mosquitoes. While suggesting that dietary routing is an important mechanism in mosquitoes, The analysis revealed that diet has a greater influence on fatty acid profiles than gender. Now pay attention please. An. The stoichiometriccentric analysis suggested that An. These findings are in line with new paradigms about quality/quantity problems in ecology, that shift away from a biomass density variable to include a two state paradigm, that represents populations or groups in a food web in regards to both their quality and quantity. Eventually, given the simplicity and rapidity of the sample analysis described herein, we suggest that these methods could have been useful to further test the models presented by Getz and Owen on a logistically feasible scale. These results lay out an experimental foundation on which to conduct future research both in the field and laboratory and may explain why increased malaria incidences was observed and reported in areas with higher inorganic fertiliser usage,.

Conceived and designed the experiments.

RHN JG CS. Performed the experiments. RHN BS CS SS OM MA MW JG. Analyzed the data. RHN BS CS SS OM MA MW JG. Contributed reagents/materials/analysis tools. Therefore, rHN BS CS SS OM MA MW JG. Wrote the paper. It is rHN CS. Conceived and designed the experiments. RHN JG CS. As a result, performed the experiments. Notice, rHN BS CS SS OM MA MW JG. Analyzed the data. Remember, rHN BS CS SS OM MA MW JG. Contributed reagents/materials/analysis tools. RHN BS CS SS OM MA MW JG. Wrote the paper. RHN CS.

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