Progress 08/15/16 to 04/14/17
Outputs
Target Audience:Imported red fire ants currently inhabit all or parts of Alabama, Arkansas, California, Florida, Georgia, Louisiana, Mississippi, New Mexico, North Carolina, Oklahoma, Puerto Rico, South Carolina, Tennessee, Texas, and Virginia. They are discovered sporadically in Kentucky, Missouri, and Maryland. Experts estimate that they spread at the rate of 5 - 20 miles per year. They have begun to spread westward into New Mexico and California, with the potential to spread up the coast to the state of Washington. The sectors most impacted are residential households and agriculture. Red fire ants have no natural enemies in the U.S. so their spread is controlled only by winter freezes and by a quarantine. In response to the invasion of red fire ants, a broad range of pesticides have been developed that are designed to control the spread of these insect pests. There are three main application methods - drench, broadcast application of on-contact pesticide, and bait. The main deficiency of chemical pesticides is their potential impact on human health. Our target audience is those seeking an effective organic alternative for invasive fire ant control primarily for an increasingly organic-oriented consumer base and for appropriate sectors of commercial organic farming and animal production. Outreach began through agricultural extension services in the affected states, particularly Alabama, Texas and Georgia and contacts with pesticide producing companies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Results: Brief Summary Previous research indicates that males store μg quantities of acetyl and hexanoyl tyramides in their external genitalia, and that they transfer these compounds to MFAs (Mature Female Alates) during the mating process. These observations led to the hypothesis that MFAs have enzymes in their reproductive system that hydrolyze tyramides to tyramine, which then passes into the now NMQ's (Newly Mated Queen) hemolymph to act on tyramine receptors. The experiments conducted confirmed that the tyramides were being converted to tyramine (via enzymatic hydrolysis) and the temperatures from 20 to 42 °C defined the temperature dependence of the enzyme that was specific to the MFA reproductive system. We also hypothesize that the male derived tyramides and/or their NMQ hydrolysis product, tyramine, are the causative agent(s) that initiate wing loss and the associated physiological changes that occur in NMQs. Injection of Tyramine or Acetyl Tyramide caused significantly faster and inappropriate dealation (not caused by the mating process) in female alates, when compared to the control. Injection of tyramine and acetyl tyramide, but not hexanoyl tyramide into MFA's also caused significant ovariole development compared to controls, in addition, injection initiated inappropriate egg-laying by the treated female alates, sometimes coupled with dealation and sometimes independent of dealation. Within the context of a colony this would be in direct conflict with the colony queen. Eggs laid would not be fertile and therefore develop into males. Feeding of tyramine and tyramides also support our above observations and importantly confirms that workers are feeding the treatments to the female alates, which in turn stimulates inappropriate dealation and significant ovariole development. The feeding experiment also demonstrates that the treatments survive the female alate gut enzymes and move into the hemolymph to act similarly to the injection experiments. Research conducted during SBIR Phase I of this project also generated experimental evidence showing, most importantly, that feeding tyramine to fire ant workers causes mortality over a range of concentrations (two orders of magnitude), which is important because the act of distributing the tyramine/sucrose solution to other workers, brood, queen, and other castes (female and male winged sexuals) dilutes the bait formulation. Experimentally, feeding 4th instar larvae caused death of those larvae. We also have evidence that workers fed the tyramine to female sexuals, causing significant pre-mature loss of their wings (within the colony, dealation) and inappropriate ovariole development. Premature wing loss, which also decreases fire ant reproductive potential, since these female sexuals will not participate in a mating flight! Dealated female sexuals directly conflict with resources going to the colony queen and multiple dealates behave as queens and act to decrease the fecundity of the real colony queen. Detailed quantitative results have been compiled and are available on request. We were unable to insert the graphs and explanations of the results of the graphs within the format and space restrictions of this report form.
Publications
|
Progress 08/15/16 to 04/14/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?PI attended the 2016 XXV International Congress of Entomology, Orlando, FL September 25-30. This meeting was held in conjunction with several other international entomology related conferences, e.g. The Entomological Society of America. This event brings together entomologists from around the world to formally and informally learn of the latest developments in the many rapidly changing areas of entomology, including basic and applied research. Contacts made and ideas generated will be beneficial in attaining the goals of this SBIR Phase I grant. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We will complete the areas reported above that are still in progress. In addition, we will investigate Sub-objective 1.2. Determine if artificial introduction of the tyramides into mature female sexual reproductive system induces wing loss and/or other NMQ physiological changes. The most difficult part of this sub-objective is development of the methodology. Success here will have spin-off into multiple other potential projects.
Impacts
What was accomplished under these goals?
Sub-objective 1.1: Water or saline solutions of each of the two tyramides and appropriate water or saline controls were prepared and injected into the abdomen of mature female alates (>15 mg). We monitored for mortality, wing loss, and ovariole development. There was little mortality. Wing loss was observed, but not as quickly as in newly mated female alates. Injection of the treatments causes significantly faster and inappropriate dealation (not caused by mating process) in female alates, when compared to the control.Eggs laid by the tyramide treated alates were higher than the controls (some times coupled with dealation and some times independent of dealation. Examination of ovariole development in the tyramide treatments showed greater development in the tyramide treatments versus the controls. Within the context of a colony this would be in direct conflict with the colony queen. Eggs laid would not be fertile and therefore develop into males. Queen pheromone is also produced by dealates, which would act to reduce the fecundity of the colony queen. For example, in polygyne colonies the higher the number of queens the lower is the mean egg-laying rate/queen. This is due to excess queen pheromone.We are continuing to fine-tune these experiments. Injection of an extract of the entire male reproductive system into mature female alates resulted in the death of the alates. We then extracted two distinguishable parts of the male reproductive system, the accessory glands and the external genitalia (source of the tyramides). Injection of the two extracts into mature female alates resulted in alate mortality only in the external genitalia extract. Control injects had no mortality during the same time period. We are continuing research to determine the cause of the mortality. Sub-objective 2.1:We have demonstrated the effects of injection of tyramides and tyramine on MFAs, but for commercial development the test compounds must show phenotypic effects through feeding, as would be applied to baits. The experimental unit for these experiments were queenright sub-colonies (queen (polygyne), brood and workers, 3 replicates). Feeding the colonies with tyramides accumulated worker mortality 9 days after being allowed to feed on 10% sucrose solutions of thetreatments. Surprisingly high worker mortality was observed from feeding workers each of the three treatments (the three treatments were not significantly different from each other). However, all of the treatments were significantly different from the sucrose controls (t-test, unpaired, parametric, 2-tailed). The starting number of workers was about 300, therefore in some replicates very few workers were left to tend the queen and brood. In view of these results we explored the worker mortality phenomenon further. To determine how quickly the our treatments and its derivatives act, we allowed 60 workers to feed on different treatments (20 in each, 3 replicates for each treatment) dissolved in 10% sucrose solution. The control and treatments were presented to the worker ants as droplets on phase separation filter paper. Consumption of the droplets was readily observed. Mortality was monitored for each of the first 10 hours and then at 24 hours. As seen here,the investigated treatments had significantly greater worker mortality than the Sucrose control (Kaplan-Meier mortality curve, Log-rank, Mantel-Cox test). Ttreatments almost had>50%mortality, respectively within the 24 h time period of the experiment. The rapid mortality and potential abnormal behavior before death suggested that the fed workers might not distribute the ingested sucrose solution to their nestmates as would normally be the case. This distribution is a cornerstone of pest ant baits. To test this, a similar experiment was conducted where workers were allowed to feed on the treatment and control solutions and were then transferred to another container with unfed nestmates. In all cases, the treatment fed workers regurgitated the treatment and sucrose solutions to their unfed nestmatesthrough the classic mechanism of trophallaxis. Thus, normal worker behavior, necessary for baitdevelopment, was not affected by the treatments under these experimental conditions. We are currently carrying out additional experiments, e.g., determination of effectiveness on larvae, effective concentrations, formulations, laboratory full colony experiments, and species-specificity.
Publications
|