The Pennsylvania State University. The Graduate School. College of Agricultural Sciences SPECIES-SPECIFICITY OF THREE COMMONLY USED AND TWO NOVEL

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1 The Pennsylvania State University The Graduate School College of gricultural Sciences SPECIES-SPECIFICITY OF THREE COMMONLY USED ND TWO NOVEL MOSQUITO FIELD-SMPLING DEVICES Thesis in Entomology by Loyal Philip Hall 2012 Loyal Philip Hall Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science May 2012

2 ii The thesis of Loyal Philip Hall was reviewed and approved* by the following: Gary Felton Professor and Department Head of Entomology Thomas Baker Distinguished Professor of Entomology Thesis dvisor James Marden Professor of Biology Michael Saunders Professor of Entomology Matthew Thomas Professor of Entomology *Signatures are on file in the Graduate School.

3 iii bstract Effective sampling is a stepping-stone to efficient use of resources, targeted control efforts, and success in nuisance or vector mosquito management. Effective sampling to identify locations where disease-vectoring mosquitoes are present and to monitor population levels allows control measures to be targeted towards medically important mosquitoes, and can reduce the environmental and financial costs associated with widespread, indiscriminate pesticide application while also preventing the failure to initiate control in an area due to a perception that there are few important mosquitoes present. comparative study between the CDC light trap, BC light trap, Reiter-Cummings gravid trap, and two traps developed by the author was conducted to test for species-specificity of each trap type. It was found that while no trap was superior over-all, certain species of mosquitoes are more likely to be detected and their populations monitored by some types of traps compared to others and the novel traps were shown to often be as effective in sampling certain important target species of mosquito as the tested commercial mosquito traps. s with the other devices, for some species the novel traps were superior and for others they appeared to be a less effective sampling device. For example, Co. perturbans tended to prefer CDC light traps, Cx. salinarius tended to prefer Hall light traps, and Cx. pipiens tended to prefer Hall gravid and Reiters-Cummings gravid traps over the other traps included in the study. 14 different species were analyzed for trap preference; results were also analyzed for WNV-infections and variety of species.

4 iv Contents List of Figures List of Tables List of bbreviations cknowledgements v vii viii ix Chapter 1, n introduction to mosquito sampling 1 Chapter 2. Species specificity of three commonly used mosquito sampling devices and two novel devices in the field. 9 Introduction 9 Materials and Methods 10 Results 18 Discussion 36 References 43 ppendix : Number of Mosquitoes In Each Trap Type By Species 46 ppendix B: Minitab Output of Statistical Tests 47

5 v Figures Chapter 1. Figure 1. Larvae. Figure 2. Pupae. Figure 3. Standard dipper Figure 4. New Jersey light trap Figure 5. BC and CDC light traps Figure 6. Reiter-Cummings gravid trap Chapter 2. Figure 7. Hall trap base Figure 8. Hall light trap Figure 9. Hall gravid trap Figure 10. BC light trap Figure 11. CDC light trap Figure 12. RC gravid trap Figure 13. typical set-up of a randomized complete block Figure 14. Histogram of the number of individuals of each species caught at a wastewater treatment plant site using the data from all 5 trap-types combined Figure 15. Histogram of the number of individuals of each species caught at a human-made wetland site using the data from all 5 trap-types combined Figure 16. Histogram of the number of individuals of each species caught at a wooded site prone to flooding using the data from all 5 traps combined Figure 17. Mean number of Culex pipiens captured per trap in the five different trap-types Figure 18. Mean number of Culex restuans captured per trap in the five different trap types Figure 19. Mean number of Culex salinarius captured per trap in the five different trap types Figure 20. Mean number of WNV-positive sub-samples per trap in the five different traptypes Figure 21. Mean number of Ochlerotatus trivitattus captured per trap in the five different trap-types Figure 22. Mean number of edes vexans captured per trap in the five different trap-types Figure 23. Mean number of Psorophora ferox captured per trap in the five different traptypes Figure 24. Mean number of nopheles quadrimaculatus captured per trap in the five different trap-types Figure 25. Mean number of Coquillettidia perturbans captured per trap in the five different trap-types Figure 26. Mean number of nopheles punctipennis captured per trap in the five different trap-types Figure 27. Mean number of Ochlerotatus canadensis captured per trap in the five different trap-types Figure 28. Mean number of Ochlerotatus japonicus captured per trap in the five different trap-types Figure 29. Mean number of Ochlerotatus triseriatus captured per trap in the five different trap-types Figure 30. Mean number of Psorophora columbiae captured per trap in the five different trap-types

6 Figure 31. Mean number of Psorophora horrida captured per trap in the five different traptypes. Figure 32. Mean number of mosquitoes, for all species combined, captured per trap in the five different trap-types Figure 33. Mean number of different species captured per trap in the five different trap-types Figure 34. Mean number of species (non- Culex pipiens/restuans) captured per trap in the five different trap-types vi

7 vii Tables Table 1. Number of mosquitoes in each trap type by species Table 2. Number of mosquitoes in each trap type by species B Table 3. Number of mosquitoes in each trap type by species C Table 4. Raw two-way NOV test results.

8 viii bbreviations BC e. n. CDC Co. Cx. EEE Och. Ps. RC WNV merican Biophysics Corporation edes nopheles Centers for Disease Control Coquillettidia Culex Eastern Equine Encephalitis Ochlerotatus Psorophora Reiters-Cummings West Nile Virus

9 ix cknowledgements. The Pennsylvania West Nile Program was instrumental to the success of this study. Their laboratory performed all of the identifications with the highest levels of skill and professionalism. The management allowed the use of this valuable Commonwealth resource because they saw the value in this study and the benefit it could bring to disease management and making their own program more effective at protecting human and animal health.

10 1 Chapter 1. n overview concerning mosquito sampling. Mosquito-vectored diseases, including malaria, yellow fever, eastern equine encephalitis, and West Nile virus, among others, kill millions of people each year and sicken hundreds of millions more. These numbers make mosquitoes the deadliest animals on earth. ccording to the Centers for Disease Control, a major priority in combating mosquito-vectored diseases is controlling mosquito populations through integrated pest management practices that incorporate surveillance, habitat modification, and changes in human behavior, along with pesticide applications that employ the least environmentally damaging yet effective control products available (Gubler et al., 2003). Effective sampling to identify locations where disease-vectoring mosquitoes are present and to monitor population levels allows control measures to be targeted towards medically important mosquitoes, and can reduce the environmental and financial costs associated with widespread, indiscriminate pesticide application while also preventing the failure to initiate control in an area due to a perception that there are few medically important mosquitoes present. The goal when sampling mosquitoes is to develop an accurate picture of the population of important species. There may be dozens of species present in a particular area. However, only certain species are important to a mitigation program or to public health officials. This study focuses on determining the efficacy of widely used mosquito sampling devices along with experimental devices developed by the author for specific mosquito species. Pennsylvania is well suited to mosquito studies. It is on the frost line, and here the ranges of northern and southern species overlap. The geologic history of the region has resulted in a wide variety of ecosystems located in close proximity. Human development of large swaths of land has resulted in the fracturing of natural habitats, reducing predators and allowing r-

11 2 strategists, like mosquitoes, to flourish. These urbanized areas provide prime habitats for many anthropophilic species. This all results in a large variety of disease-vectoring and nuisance species in Pennsylvania, where 59 species of mosquito are recognized by the state s West Nile Virus Program. In Pennsylvania and the surrounding areas, several mosquito-vectored maladies are endemic, including West Nile virus (WNV), St. Louis encephalitis (SLE), LaCrosse encephalitis, eastern equine encephalitis (EEE), and dog heartworm. The introduction/reintroduction of other mosquito-vectored diseases is a concern of the state s WNV program with the increase in international travel and invasive species. Malaria and chikungunya have appeared in sporadic outbreaks in border states. Dengue is a concern for introduction due to the invasive species edes albopictus that is now common in many parts of Pennsylvania, although it is currently not found in Lebanon County where the trials reported herein were conducted. There are many methods for sampling mosquitoes. Because mosquitoes have an aquatic larval stage and a flying adult stage the sampling processes can be divided into adult and larval sampling techniques. Mosquito larvae are aquatic, but do breathe air. Many genera, Figure 1. Larvae. such as Culex and edes have a snorkel-like siphon on their dorsal posterior, whereas the genera nopheles simply have spiracles in the same location through which they breathe (Fig. 1). Pupae of all species have two breathing tubes located dorsally called trumpets (Fig. 2). Most Figure 2. Pupae. mosquito larvae and pupae hang on the surface tension of the water, with their breathing apparatus exposed to the air and the rest of the organism under water. Larvae and pupae are most commonly sampled while resting on the water surface using a dipper (Fig. 3), (Leisnaham

12 3 et al., 2005). Standardized dippers consist of a long telescopic or fixed handle with a 1-pint white plastic cup affixed to the end (Hagstrum, 1971). Techniques for using a dipper include scooping out larvae, placing the dipper next to larvae to allow them to flow into the cup, or scraping it against vegetation or the substrate on which larvae may be sheltering. The larvae can be removed into collection vials by pouring them out or, more commonly, by pipetting them into the vials. nother strategy is to use smaller, non-standard dippers, such as a white kitchen ladle, to sample larvae from narrower containers like discarded tires. For even narrower openings, such as tree-holes or human refuse, a large pipette (a turkey baster is common) can be used. The Figure 3. Standard dipper. larvae of one particular species, Coquillettidia perturbans, insert their siphons into the stems of aquatic plants, using them as extended snorkels, and must be collected by pulling up the plants and agitating them in a container that is then inspected for the larvae. The number of larvae and species content gathered by dipping is highly dependent upon operator skill and technique used (Miura et al., 1970; Hagstrum, 1971; Leisnham, 2005). Certain species habits make them more conspicuous than others, whereas others will hide out on the bottom for a long time when disturbed, out of sight and uncaught while breathing subcutaneously. Early instars can easily be missed due to their size and lack of contrast in the dipper. For all these techniques, the larvae and their medium can be transferred to white sorting pans to increase contrast and thus increase detection capabilities. mong the strategies for collecting adult mosquitoes are aspiration, landing counts, and trapping. dult mosquitoes do fly, but most species are very weak flyers and unless actively seeking some resource, they prefer stay at rest in some sort of sheltered environment such as

13 4 vegetation or on the walls of a building. Mosquitoes at rest can be collected by aspiration (Chen et al., 2011). However aspiration of mosquitoes at rest is labor intensive and requires relatively quiescent mosquitoes. Sampling adult mosquitoes can be performed most easily by offering some resource that serves as an attractant. The oldest of these techniques is a landing count survey in which humans offer themselves as the attractant. The number of mosquitoes landing in a specified period of time can be counted and aspirators can be used to collect the mosquitoes landing on the person for identification or testing, when called for. Landing count surveys work well for anthropophilic species that are host-seeking (Ndiath et al., 2011; Hiwat et al., 2011). However, not all species are so inclined to feed primarily on humans, and humans differ between individuals in mosquito attractiveness (Costantini et al., 1997; Lefevre et al., 2010). There is often a short time-window during which host-seeking behavior is at its zenith, which greatly limits the number of samples that can be taken by an individual or team. Other concerns are possible health risks and violations of worker protection laws (Ndiath et al., 2011). Trapping mosquitoes lured to an effective attractant has become increasingly popular because it is costand labor-effective and traps can sample the less anthropophilic species that often play key roles in disease cycles. One of the earliest and most well known traps is the New Jersey light trap (Reinert, 1989) (Fig. 4). These traps are baited using a light bulb, and a fan then directs the insects into a collection chamber containing a killing agent. The trap is Figure 4. New Jersey Light Trap.

14 5 powered with an C electric source. These traps are still in use today. Their fixed locations and constant collections allow for consistent, long-term data collection as well as pre- and postpesticide application sampling without the variability introduced by changing trap locations. Many species can be well sampled using light as an attractant. However, some species are not, and many other non-target insects besides mosquitoes are collected and confound the processing of the samples (Reinert, 1989). The traps are also not portable and require C current. CO 2 -baited traps are also commonly called light traps because they are based on the New Jersey light trap design and were historically baited with a light source (Derraik et al., 2010). DC current (batteries) powers the fan that creates air-flow to draw mosquitoes into a collection chamber, without a killing agent. These features allow for greater portability but also make the traps vulnerable to theft and battery failure. The later introduction of a no-kill collection chamber allowed for better disease detection. CO 2 was also added later as an additional attractant (Reeves, 2001). It was found that by removing the light and using solely CO 2 the traps collected a cleaner sample, i.e., having a larger portion of the sample consisting of mosquitoes and a lower percentage of non-target insects. lthough the light is Figure 5. BC (left) and CDC light traps. still an option for most light traps, it is commonly turned off. Two of the more common CO 2 - baited light traps are the BC and the CDC light traps (Fig. 5). Dry ice or CO 2 compressed gas tanks are the most common sources of CO 2. There are transportation and supply logistics for both options. Dry ice is the most common CO 2 form, due to its portability and availability, in well populated, developed regions. Compressed-gas CO 2 tanks are more common when dry ice is not feasible or when CO 2 flow rates need to be very accurately controlled (Jawara et al., 2011). With dry ice, there is the issue of quality. Dry ice

15 6 can be of different forms, ranging from powdery, loosely packed blocks, to hard pellets, or else very dense, solid blocks. These different forms of dry ice sublimate at faster or slower rates, respectively, creating highly variable CO 2 emission rates and confounding trap performance. Gravid traps (Fig. 6) are those that attract ovipositing female mosquitoes. These are baited with an infusion, consisting of hay, grass, or other vegetation that has been allowed to decompose in water for several days or weeks, imbuing the water with bacteria, decomposing organic matter and volatile emissions that are attractive to the females of many species. The infused water can thus function as an effective bait for this type of trap. The emitted volatiles Figure 6. Reiter- Cummings gravid trap. attract gravid females looking for quality larval habitat in which to lay eggs. The mosquito species that are attracted are limited to those that lay eggs directly in water rather than those that lay eggs on a dry substrate that is likely to be flooded later (Chen et al 2010). DC-powered motorized fan draws the females that have arrived to investigate the bait into a collection chamber or net. The battery power for gravid traps imposes the same benefits and drawbacks as light traps, including issues of portability, theft, and battery failure. Despite the term hay infusion, there can be quite a bit of variability in the ingredients used along with duration of brewing, pre-existing bacterial culture, ambient temperature, sunlight, air flow, etc. (Ponnusamy et al., 2009; McPhatter et al., 2009). The creation of the infusion varies, and each person or program often has their own recipe. The standard procedure for Pennsylvania s West Nile program is to stuff a large mesh bag with straw, which is then placed in a 65 gallon black trash can, filled with water and a small amount of milk or milk albumin, and then left in a sunlit area for at least a week. The vegetation used to create the infusion does influence which species are likely to be caught, and many tree-hole species such as

16 7 Och. triseriatus and Och. hendersoni will avoid the highly organic straw infusions, preferring an oak leaf infusion (Trexler et al., 1998). The amount of infusion used influences sampling efficacy. study by Michael Hutchinson presented at the 2010 Mid-tlantic Mosquito ssociation Meeting (unpublished) showed that the distance between the water surface and the sample intake pipe, which is a result of using greater or lesser amounts of infusion, directly affected the number of mosquitoes collected (i.e. the greater the distance the greater the number of mosquitoes captured). Other traps have been created as a result of an inability to collect important diseasetransmitting species using any of the above types of traps. edes albopictus, a denguetransmitting and highly anthropophilic species is notoriously disinclined to being sampled by customary traps (Barnard et al., 2011; Farajollahi et al., 2009). For many years, landing counts and larval surveys were the only way to somewhat reliably sample this important mosquito. Of the several novel types of traps created to sample this species, the most well known is the BG Sentinel, which uses a lure containing 1-octen-3-ol ( octenol ) and lactic acid as a volatile lure, along with visually contrasting colors and a counterflow capturing technique, plus optional CO 2 (Rajollahi et al., 2009). Other trap-types targeting e. albopictus include the Faye-Prince and the Zuumba traps. Culiseta melanura is a bird-feeding mosquito species that serves to amplify eastern equine encephalitis in the wild. This species is rarely detected using any kind of trap. dding to the degree of difficulty, the larvae use hidden crypts beneath trees. For this and other such challenging species, resting boxes may be used, which are basically boxes with one lateral side open (Howard et al., 2011). Resting boxes provide a humid, dark, sheltered environment for mosquitoes to repose. They can be wooden or cardboard, painted, lined with cloth or unfinished (Govella et al., 2011). n operator checking the box will aspirate the resting mosquitoes from the walls or else place a cover on it and place the entire resting box into dry ice, a freezer, or use

17 8 some other method to kill or incapacitate the mosquitoes for collection. lthough this does not result in large numbers of mosquitoes being collected, compared to using a gravid trap for Culex restuans for example, it is often the only viable strategy for species such as this, despite its being labor intensive. Sampling success when using mosquito traps is highly dependent upon operator skill in trap placement and attractant quality. Proximity to refuge habitats, trap height, amount of attractant used, attractant formulation, the juxtapositioning of the dry ice container and the trap, contamination of traps with substances on the operator, and micro-habitats that create fluctuations in wind, lighting, and humidity can all influence the sampling results. This variability demands training and experience for those collecting samples in the field. Despite the inherent variability, the use of mosquito traps is still more reliable than the use of landing counts or personal observations.

18 9 Chapter 2. Species specificity of three commonly used mosquito sampling devices and two novel devices in the field. Introduction. CO 2 -baited light traps and gravid traps are two of the most common types of devices used for vector mosquito surveillance in the world. s a West Nile mitigation program manager, I have found myself usually needing to carry a full complement of both gravid and CO 2 -baited traps. The models that are commonly used are very bulky. I have developed a novel trap, a compact convertible trap that can be used as either a gravid or a CO 2 light trap. Comparative studies between multiple models of gravid traps or different models of CO 2 trap for particular species efficacies are uncommon, yet would be very useful for improving vector sampling efficacy. Hence, this need formed the impetus for the current study. ny particular pathogen is normally vectored by certain mosquito species more than others because different mosquito species have different behavior patterns and physiologies. These behavioral differences also imply that certain types of traps may be more effective than others at capturing particular species (Vaidyanathan and Edman, 1997) because of the traps various shapes, sizes, colors, airflows, vibrations, etc. (llen and Kline, 2004). For example, a particular trap may be more effective at sampling Co. perturbans (vector of eastern equine encephalitis) while another may be more effective for Cx. pipiens (vector of West Nile Virus). It is predicted that through this study, each model tested will have been shown to have different optimal efficacies for particular species, which will inform vector control specialists how to optimize their sampling protocols for different target mosquito species through the use of appropriate traps.

19 10 Materials and Methods. Because my novel devices are new to mosquito researchers, further description is warranted. I designed the Hall Trap to be a compact, convertible trap that can be used as either a CO 2 light trap or a gravid trap. The trap body is a cylinder with a fan at the exhaust end and a collection chamber between the intake and the fan (Fig. 7). The lack of fan pass-through by this technology Figure 7. Hall trap base. results in better quality samples. Black burlap cloth encircles the intake opening to create contrast and to absorb aromas, giving the exterior a more organic appearance. In light-trap mode, the trap base is hung beneath a 2- liter insulated beverage container having holes drilled in the sides that is filled with dry ice (Fig. 8). In gravid-trap mode, the trap base is placed on Figure 8. Hall light trap. its side and inserted through an opening into the side of a twogallon black plastic bucket filled to the opening with hay infusion (Fig. 9). The standard, currently commercially available light-trap and gravid-trap models that a West Nile mitigation program Figure 9. Hall gravid trap manager such as myself has heretofore used are bulky, taking up large volumes of storage space in a vehicle. The Hall Trap is convertible and compact, meaning more flexibility in the field and leaving more storage space for sprayers, pesticides, dippers, tripods, tools, BG Sentinels for e. albopictus, educational materials and other tools of the trade. When a professional such as myself is in the field, the trap, battery, and attractant can all be carried together with one hand using the bucket, allowing a free hand to carry a machete to clear

20 11 a path or a dipper for larval surveillance. The Hall Trap technology is being offered to potential manufacturers by the Penn State Intellectual Property Office. This study compared the merican Biophysics Company light trap marketed by Clarke Mosquito (i.e., the BC light trap ; Fig. 10), the Centers for Disease Control light trap marketed by BioQuip (i.e., the CDC light trap ; Fig. 11), the Reiter-Cummings gravid trap marketed by BioQuip (the RC gravid trap ; Fig. 12), the Hall light trap (Figure 8), and the Hall gravid trap (Figure 9). Using the protocols employed by Pennsylvania s West Nile Program, when operating the three commonly used traps, the light bulbs were removed from the BC and the CDC trap and the light sensor for the fan motor was covered on the Reiter-Cummings trap. Figure 10. BC light trap Figure 11. CDC light trap Figure 12. RC gravid trap Over the course of the 2-year study, traps were all stored in the same location, next to each other, when not in use. ll traps were kept in good repair, their motors cleaned and oiled on a regular basis and replaced when running roughly.

21 12 Figure 13. typical set-up of a randomized complete block. Sites for trials were chosen by selecting high mosquito population locations as ascertained from the West Nile Program s regular surveillance data. large variety of sites was required for the greatest diversity in habitat and potential mosquito species inhabiting these locations. Sites that were utilized included wastewater treatment plants, wetlands, flooding terrains, and human-discarded-container-rich sites. Thirty-nine usable blocks were conducted, with a small number of blocks having to be discarded due to battery failures, a tree falling over onto a trap, and passers-by tampering with traps.

22 13 Nineteen different sites were utilized. t each site a replicate (randomized complete block) was deployed that was comprised of the five trap-treatments evenly spaced approximately 10 m apart within a relatively homogeneous area (c.f., Fig. 13). Dice were used to randomly assign traps to a position within the block each night in order to prevent bias in trap interactions, micro-environments, researcher preferences, or other factors such as wind direction that could unduly impart bias to the treatment means within blocks. Tripods were used for hanging light traps at a uniform height (1 m) and the gravid traps were set on the ground. t the time the traps were set for each night, the dry ice chambers of the light traps were filled to capacity (5 lbs). RC gravid traps received 1 inch of hay infusion in the pan (Hutchinson, 2010) and the Hall gravidtrap ( Hall-G ) was filled to the brim of its side opening with the same hay infusion solution from the same bottle as was used for the RC gravid trap. The infusion was agitated before pouring it into either trap-type. Hay infusions were replaced in the traps each day the traps were sampled and their positions re-randomized. Once all traps were set up, batteries were connected to all the traps in a block. Upon collecting samples, collection chambers were all removed first, in the same order batteries were connected, and then the traps were taken down. Having all traps start and stop as temporally close as possible was important to reducing any possible bias due to one trap collecting mosquitoes while other traps in the block were not. Collection chambers were marked with trap data using a permanent marker as they were removed from traps. fter collection, on the same day, the chambers were placed on dry ice to kill the mosquitoes in the samples. Non-mosquitoes were then picked out of the samples and the mosquitoes were placed into vials, labeled with date, site, and trap type, and delivered on dry ice to Pennsylvania s West Nile Virus Program s laboratory to be identified to species and tested for West Nile virus by their technicians.

23 14 Testing for WNV by the P DEP Laboratory was typically performed by IgM ELIS or PCR. Samples were divided into species-specific sub-samples. Sub-samples with more than 50 mosquitoes were divided into batches of 50 mosquitoes each. Therefore, a sample from a single trap can yield multiple WNV-positive sub-samples. Multiple sub-samples testing positive for WNV is indicative of high WNV prevalence in that population. Data was compiled into a spreadsheet and then analyzed using MiniTab 14 statistical analysis software. The data included a total of 39 blocks, each block measuring the number of each species caught in each mosquito trap and the number of WNV-positive sub-samples in each trap. The data was log-transformed normalized after all zeroes were replaced by a value of 0.5. Block and treatment variances were accounted for using a 2-way nalysis of Variance, and differences among treatment means were determined using Tukey s w-procedure. The performances of the five trap types were analyzed in terms of their ability to capture different species, to capture individuals carrying the West Nile virus, to collect a wide variety of species, and to capture the greatest number of mosquitoes. It was understood that not all species were going to be present during each sampling period. For instance, species such as Och. canadensis, are known to be highly seasonal, being found in large number only at certain times of the year. Certain types of trapping habitats were anticipated to be populated with a narrower range of species than others. One example involves Psorophora horrida, which is dependent on specific weather events, including heavy flooding, to trigger the hatching of its eggs. For a practical example, the reader can refer to Figs. 14, 15 and 16, in which the species demographics from three different sites used in this study are displayed.

24 Number of mosquitoes 15 Wastewater treatment plant e. vexans Cx. pipiens Cx. restuans Cx. salinarius Och. triseriatus Mosquito species Figure 14. Histogram of the number of individuals of each species caught at a wastewater treatment plant site using the data from all 5 trap-types combined. t the wastewater treatment plant site (Fig. 14) there was a reed bed for the filtration of the wastewater. The primary species present in this location were Cx. pipiens and Cx. restuans. few Cx. salinarius were apparently able to colonize the location, or else were possibly migrants from nearby. small number of e. vexans was captured, possibly a result of nearby flooding, and Och. triseriatus likely had emerged from a tire, a bucket or some other container on-site.

25 Number of mosquitoes Wetland habitat Species Figure 15. Histogram of the number of individuals of each species caught at a human-made wetland site using the data from all 5 trap-types combined. This wetland habitat site (Fig. 15) comprises a human-made wetland used for stormwater management. It exhibited a wider species diversity than did the wastewater treatment plant site. There was a variety of microhabitats within this wetland, including permanent water, flooding terrain, and aquatic, reedy vegetation. The flooding areas under open sky would have produced the e. vexans and Och. trivitattus that were captured. The permanent water areas would have been responsible for producing the nopheles and Culex species. few Co. perturbans apparently had colonized the reedy areas.

26 Number of mosquitoes 17 Wooded, flooding terrain Species Figure 16. Histogram of the number of individuals of each species caught at a wooded site prone to flooding using the data from all 5 traps combined. The wooded, flooding terrain (Fig. 16) most certainly contained water sources that had persisted for some time, because species of both Culex and nopheles were present in abundance. Cx. restuans in particular is known to utilize woodland pools. t this site, a greater variety of floodwater species was present than in the wetland site, including e. vexans, Och. trivitattus, Ps. ferox, and Ps. horrida, all probably due to the lush tree canopy. In preparing the experimental design, therefore, it was determined that it was not appropriate to include blocks from habitats or time periods having no individuals of a particular species present. To do otherwise would provide a less accurate estimate of the true experimental error variance. Thus the design anticipated that for each species being analyzed, only blocks that had captured at least one individual of the species of interest were included in the block replication of the analysis. For example, when conducting statistical tests comparing the five

27 18 traps capture performances with regard to n. quadrimaculatus, out of the total 39 blocks there were 16 blocks in which at least one of the traps caught one individual. It was deemed according to the design that n. quadrimaculatus were only present at those 16 times and places such that the traps capture abilities in those sample blocks could be appropriately assessed. This same strategy was likewise applied to other comparisons, such as the analysis of traps abilities to detect WNV-positive mosquitoes. Results. Species-by-species trap performance. The trapping results showed that the novel trap types (Hall traps) performed as well as, and sometimes better than, the currently used, standard commercial traps for the majority of the disease-vectoring mosquito species and there were differential capture efficacies for the traps used in this study for different species. Thirty-nine replicates were obtained over two years. Histograms of the mean number of each species and WNV-positive sub-samples caught per trap, along with significance indicators are displayed in Figures Means, standard deviations of the means, and the Mini Tab outputs of the 2-way NOV with Tukey s w-procedure are displayed in Tables 1-4 in the ppendix section of this thesis. Cx. pipiens was captured in significantly higher numbers in the Hall-G and RC traps than in the other trap-types (Fig. 17). It is one of the two primary vectors of West Nile virus in the north/mid-eastern US. Interestingly, it will feed on birds early in the season and then switch to feeding on larger mammals such as humans later in the summer, making it very effective for WNV transmission to humans. Birds are the reservoir for WNV and humans are dead-end hosts. Cx. pipiens tends to have its largest populations in late summer and utilizes containers, long-term

28 Mean number of mosquitoes per trap 19 standing water, and highly organic water such as from waste-water treatment plants and farm run-off. It does not have a long flight range, being typically less than one mile. 45 Captures of Culex pipiens B 10 B 5 0 BC-L CDC-L Hall-G Hall-L RC-G Trap type B Figure 17. Mean number of Culex pipiens captured per trap in the five different trap-types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w-procedure (P < 0.05; N= 34). This species was captured predominantly near long-term standing water, waste-water treatment facilities and container-rich locations. Cx. restuans likewise was captured at significantly higher capture rates in the Hall-G and RC traps than in the other trap-types (Fig. 18). This species is the other primary vector of WNV in the north/mid-eastern US. It feeds mostly on birds, but will feed occasionally on other animals and humans and serves as an amplification vector for WNV among birds. It tends to have its peak population in spring and early summer. Cx. restuans lays eggs on long-term standing water such as ephemeral spring pools, in containers, and moderately organic water such as is found in off-line waste-water treatment plants and unused manure lagoons. Its flight range is also relatively short, typically less than a mile.

29 Mean number of mosquitoes per trap 20 Captures of Culex restuans B 20 B B 0 BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 18. Mean number of Culex restuans captured per trap in the five different trap types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w-procedure (P < 0.05; N= 35). This species was captured predominantly near long-term standing water, waste-water treatment facilities and container-rich locations. Cx. salinarius exhibited the highest levels of capture in the Hall-L trap, which outperformed the BC, RC and Hall-G traps (Fig. 19). The CDC trap outperformed the 2 gravid traps (Fig. 19). Cx. salinarius is a major West Nile virus bridge vector, feeding readily on humans and birds. This species larvae are found primarily in mildly brackish waters, such as coastal wetlands and reclaimed salt-marsh habitats. However, it can utilize the same habitats as Cx. restuans, but with much less success. Phosphates can also create favorable larval conditions (Slaff and Haeffner, 1985) and so an agricultural field that has been fertilized and subsequently flooded for several weeks or storm run-off from a fertilized lawn has the potential to become an extremely productive habitat hundreds of miles from the nearest naturally occurring brackish water.

30 Mean number of mosquitoes per trap 21 Captures of Culex salinarius B 4 BC 2 C C 0 BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 19. Mean number of Culex salinarius captured per trap in the five different trap types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w-procedure (P < 0.05; N= 23). This species was captured predominantly near long-term standing water locations. In the 8 blocks where WNV was detected in the mosquitoes, the RC (Reiter-Cummings) gravid trap captured a significantly greater number of WNV-positive mosquitoes than the Hall-G gravid trap, which also outperformed the other trap-types in this regard (Fig. 20). Because the vast majority of WNV positive samples detected since the implementation of regular surveillance in P have been either Cx. pipiens or Cx. restuans, it is not surprising that the gravid traps, which were most successful with these species (Figs. 17, 18), should also be most successful at detecting the presence of this virus in mosquitoes.

31 Mean number of species per trap 22 Number of West Nile-positive sub-samples B C C C 0 BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 20. Mean number of WNV-positive sub-samples per trap in the five different trap-types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w-procedure (P < 0.05; N= 8). These positive samples were found exclusively in Cx. pipiens and Cx. restuans; other species were also tested but WNV was not detected in those other species.

32 Mean number of mosquitoes per trap 23 Och. trivitattus was captured in statistically insignificantly different numbers by the Hall- L, the CDC and the BC traps. ll three of these light trap types were more effective than the two gravid traps (Fig. 21). This is a floodwater species that lays its eggs on dry land, the larvae hatching after being flooded. The larvae are very wary and will spend long periods of time submerged when disturbed. The mosquito adults are major human pests, and are a bridge vector of WNV and dog heartworm 25 Captures of Ochlerotatus trivitattus B B 0 BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 21. Mean number of Ochlerotatus trivitattus captured per trap in the five different trap-types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w- procedure (P < 0.05; N= 15). This species was captured predominantly near flooding terrain.

33 Mean number of mosquitoes per trap 24 e. vexans was captured at as high a rate in the Hall-L traps as for the BC and CDC traps (Fig. 22). ll these light traps captured significantly greater numbers of this species than the RC and Hall-G gravid traps. This is a floodwater species, laying its eggs on dry land and then hatching following flooding. e. vexans does not require the water to be present for long before the eggs hatch. The adults have a relatively long flight range, often being found several miles from where they emerge from their pupae. It is a common vector of dog heartworm and a bridge vector for West Nile virus and EEE. Captures of edes vexans B B BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 22. Mean number of edes vexans captured per trap in the five different trap-types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w-procedure (P < 0.05; N= 30). This species was captured predominantly at sites near flooding terrain.

34 Mean number of mosquitoes per trap 25 In some cases, the commercial traps performed better than the novel trap-types. One such instance occurred in trapping Ps. ferox, another floodwater species. The adults prefer wooded habitats and can be a bridge vector for WNV. Only 6 blocks contained traps that captured this species, and the CDC and BC traps outperformed the RC and Hall-G traps. (Fig. 23) Captures of Psorophora ferox B BCD D CD BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 23. Mean number of Psorophora ferox captured per trap in the five different trap-types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w- procedure (P < 0.05; N= 6). This species was captured predominantly near flooding terrain.

35 Mean number of mosquitoes per trap 26 nother case of commercial traps outperforming the novel trap-types occurred in the trapping of n. quadrimaculatus, a vector for human malaria and a bridge vector for WNV and EEE. dults of this species were captured in the CDC trap at significantly higher numbers than in other trap-types (Fig. 24). n. quadrimaculatus has a wide range of habitats. The larvae thrive in clean, clear water such as pristine wetlands, but they can also be found in containers, off-line wastewater treatment plant tanks, and at the edges of moving water. Captures of nopheles quadrimaculatus B 4 2 B B B 0 BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 24. Mean number of nopheles quadrimaculatus captured per trap in the five different trap-types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w- procedure (P < 0.05; N= 16). This species was captured predominantly near wetland and container-rich locations.

36 Mean number of mosquitoes per trap 27 Co. perturbans, a vector for Eastern Equine Encephalitis, also was captured in significantly greater numbers by the CDC trap than in other traps (Fig. 25). Eastern Equine Encephalitis is a deadly, though rare, disease that is amplified by another species, Culesita melanura, which is limited to acidic (usually red maple) wetlands, through birds and then Cq. perturbans (and a few other species) acts as a bridge vector, transmitting the disease to humans. The larvae of Cq. perturbans are found in clean water with reedy plants. The larvae insert their siphons into the stems of those plants, effectively using them as snorkels, allowing the larvae to stay submerged indefinitely and making them difficult to detect. 3 Captures of Coquillettidia perturbans B B B B BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 25. Mean number of Coquillettidia perturbans captured per trap in the five different trap-types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w- procedure (P < 0.05; N= 10). This species was captured predominantly near wetland locations.

37 Mean number of mosquitoes per trap 28 n. punctipennis adults were captured at insignificantly different levels by all five types of traps (Fig. 26). This species has the ability to transmit human malaria and its larvae thrive in clean water habitats, including stream edges, wetlands, and ephemeral pools. The number of blocks in which at least one adult was captured (14) would seem to have been sufficient to assess the differential capture rates of the traps, yet no such differences arose, even between the gravid traps and the light traps (Fig. 26). 1.8 Captures of nopheles punctipennis BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 26. Mean number of nopheles punctipennis captured per trap in the five different trap-types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w- procedure (P < 0.05; N= 14). This species was captured predominantly at sites near wetlands.

38 Mean number of mosquitoes per trap 29 Several other species were captured in numbers of blocks that were apparently too low to allow a determination of any significant differences among the trap types capture abilities. Och. canadensis is an example of this, and is a very early season species. The larvae can be found in ephemeral pools formed by snow melt, and even in melt water in the snow. It is a bridge vector for EEE. Only two blocks contained traps that captured individuals of this species, with all of them being light traps (Fig. 27). 4.5 Captures of Ochlerotatus canadensis BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 27. Mean number of Ochlerotatus canadensis captured per trap in the five different trap-types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w- procedure (P < 0.05; N= 2). This species was captured predominantly near wooded areas with early spring ephemeral pools.

39 Mean number of mosquitoes per trap 30 nother example is Och. japonicus, which is an invasive species that was first detected in the US in 1998, and whose larvae utilize containers. It is an efficient vector of WNV. Only 7 blocks contained traps that captured individuals of this species, all of them being gravid traps (Fig. 28). 3.5 Captures of Ochlerotatus japonicus BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 28. Mean number of Ochlerotatus japonicus captured per trap in the five different trap-types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w- procedure (P < 0.05; N= 7). This species was captured predominantly near container-rich sites.

40 Mean number of mosquitoes per trap 31 Och. triseriatus (Fig. 29) is a container-utilizing mosquito species. It normally lays eggs in tree holes, but will also use tires and other artificial containers. It prefers larval habitats with low organic loads, usually opting for water with only decaying leaves to provide sustenance for the larvae. This species is a vector for St. Louis Encephalis (SLE) and can be a bridge vector for WNV. Only five blocks contained traps that captured this species, comprising a mixture of gravid traps and light traps. Captures of Ochlerotatus triseriatus BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 29. Mean number of Ochlerotatus triseriatus captured per trap in the five different trap-types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w- procedure (P < 0.05; N= 5). This species was captured predominantly near areas with containers.

41 Mean number of mosquitoes per trap 32 Only 6 blocks contained traps that captured Ps. columbiae (Fig. 30), with all five traptypes contributing to mosquito captures. It is a floodwater mosquito and can occur in great numbers and act as aggressive pests of humans and cattle. This species also can be a bridge vector for WNV. 18 Captures of Psorophora columbiae BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 30. Mean number of Psorophora columbiae captured per trap in the five different trap-types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w- procedure (P < 0.05; N= 6). This species was captured predominantly near flooding terrain.

42 Mean number of mosquitoes per trap 33 Ps. horrida is a relatively rare woodland floodwater species. It is similar to Ps. ferox in both habitat and morphology. In the entire state of Pennsylvania only 27 traps since 2001 from the P WNV Program have managed to trap individuals of this species. In the current study, however, three of the light-traps from two different blocks did manage to capture these rare mosquitoes, now making a total of 30 trap-capture detections (Fig. 31). 6 Captures of Psorophora horrida BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 31. Mean number of Psorophora horrida captured per trap in the five different trap-types. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w- procedure (P < 0.05; N= 2). This species was captured predominantly near flooding terrain. cross-species trap-type performances. It is well known that gravid traps can capture very large numbers of mosquitoes, particularly Cx. pipiens and Cx. restuans. Capturing over 1000 in a gravid trap, although not common, is not highly unusual. lthough while not in the course of conducting this study, rather

43 Mean number of mosquitoes per trap 34 during regular surveillance for WNV in 2011, an RC gravid trap captured 2,578 mosquitoes and a Hall-G captured 2,275 mosquitoes. Out of the 541 gravid traps deployed in Lebanon County, P in 2011, 10 had catches over Extraordinarily high populations of mosquitoes are necessary to capture more than a few hundred mosquitoes in a light trap. Thus it was expected that the gravid traps should outperform the light traps over-all (Fig. 32). 160 Captures of all species combined B B B 20 0 BC-L CDC-L Hall-G Hall-L RC-G Trap type Figure 32. Mean number of mosquitoes, for all species combined, captured per trap in the five different traptypes. Means having no letters in common are statistically different according to a 2-way NOV followed by Tukey's w-procedure (P < 0.05; N= 39). In terms of variety of species caught, the gravid traps caught, on average, the greatest variety (Fig. 33). However, I believe this to be because they were extremely effective at catching Cx. pipiens and Cx. restuans. These mosquitoes were ubiquitous, being found together in almost every block (Figs. 17, 18). The other species were not so common, only e. vexans being found in more than half of the blocks. The gravid traps were not very effective at catching