REPRODUCTIVE STRATEGY of Pheropsophus Aequinoctialis L

Home , Lebia chlorocephala, Ormia depleta, Pheropsophus, Scapteriscus, Scapteriscus abbreviatus

REPRODUCTIVE STRATEGY OF Pheropsophus aequinoctialis L.: FECUNDITY, FERTILITY, AND OVIPOSITION BEHAVIOR; AND INFLUENCE OF MOLE CRICKET EGG CHAMBER DEPTH ON LARVAL SURVIVAL

AARON SCOTT WEED

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2003

Aaron Scott Weed

This thesis is dedicated to my family and my closest friends who have inspired me, given me confidence, and had faith in me throughout this experience.

ACKNOWLEDGMENTS

I would first like to thank my committee members (Drs. Howard Frank, James

Nation, and Heather McAuslane) for their instructional criticisms concerning this thesis.

Secondly, I would like to thank all of my fellow graduate students for their scientific input and friendship; and for sharing this educational experience with me. Many thanks go to Robert Hemenway, who provided me with all of the necessary tools and insects to make this thesis possible. I would sincerely like to thank Marinela Capanu of IFAS

Statistics for her help with all of the statistical analyses. Big thanks go to Alejandro

Arevalo, for the use of his camera; and to him and Craig Welch, for their companionship in Dr. Frank’s laboratory. Finally, I would like to thank Debbie Hall for all of her guidance throughout this degree process.

TABLE OF CONTENTS Page

ACKNOWLEDGMENTS...... iv

LIST OF TABLES ...... vii

LIST OF FIGURES...... viii

ABSTRACT……………………………………………………………………………….x

CHAPTER

1 REVIEW OF LITERATURE...... 1

Mole Cricket Diversity in the Southeastern United States ...... 1 Mole Cricket Biology...... 1 Mole Cricket Damage ...... 3 Control of Pest Mole Crickets...... 4 Chemical Control of Mole Crickets...... 5 Cultural Control to Prevent Mole Cricket Damage ...... 7 Biological Control of Mole Crickets in Florida...... 7 Carabid Beetle Ecology ...... 11 Tribe Peleciini……...... 13 Tribe Lebiini………...... 14 Tribe Brachinini……...... 15 Objectives……...... 17

2 FECUNDITY AND FERTILITY OF LABORATORY-REARED Pheropsophus aequinoctialis...... 19

Introduction…...... 19 Materials and Methods...... 20 Experimental Colony ...... 20 Egg Collection and Fertility Determination...... 20 Results………...... 21 Discussion…… ...... 22

v 3 OVIPOSITION BEHAVIOR OF Pheropsophus aequinoctialis L. IN RESPONSE TO SAND WITH Scapteriscus abbreviatus SCUDDER TUNNELS, ARTIFICIALLY CREATED TUNNELS, AND TO SAND WITHOUT TUNNELS: DIRECTED OR RANDOM? ...... 28

Introduction…...... 28 Materials and Methods...... 29 General Plexiglas Sandwich ...... 29 Mole Cricket Tunnel (MCT) versus No Tunnel (NT) ...... 30 Mole Cricket Tunnel (MCT) versus Artificial Tunnel (AT) ...... 30 Artificial Tunnel (AT) versus No Tunnel (NT)...... 31 Oviposition…………...... 32 Sampling Sandwiches with Treatments...... 32 Egg Extraction……… ...... 32 Results………...... 34 MCT versus NT……...... 34 MCT versus AT…… ...... 35 AT versus NT………...... 35 Discussion…… ...... 36

4 INFLUENCE OF Scapteriscus abbreviatus EGG CLUTCH DEPTH ON THE LARVAL SURVIVAL OF Pheropsophus aequinoctialis...... 44

Introduction…...... 44 Materials and Methods...... 45 Scapteriscus abbreviatus Egg Chamber ...... 45 Placing Egg Chambers over a Range of Depths ...... 46 Results………...... 47 Discussion……...... 47

5 CONCLUSIONS AND FUTURE RESEARCH...... 51

APPENDIX ADDITIONAL CHAPTER 2 FIGURES ...... 54

LIST OF REFERENCES ...... 59

BIOGRAPHICAL SKETCH...... 64

LIST OF TABLES

Table page

2-1 Emergence dates (2002) of the female and 2 males placed in each of the 10 cups from which eggs were collected...... 25

3-1 Results of the contrasts procedure between depth intervals for both treatments ..... 38

vii LIST OF FIGURES

Figure page

2-1 Mean number of eggs laid per week with 95% confidence intervals for each female ...... 26

2-2 Fecundity versus the proportion of fertile eggs laid for each female over the 20-week period ...... 26

2-3 Mean number of eggs laid per week with 95% confidence intervals for all females...... 27

3-1 Plexiglas sandwich with black construction paper in place...... 39

3-2 No tunnel (NT) and Mole cricket tunnel (MCT) treatments with Plexiglas barrier in place ...... 39

3-3 Placement of the artificial tunnel (AT) treatment in all oviposition arenas...... 39

3-4 General MCT versus AT arena before the beetle was released for oviposition ...... 40

3-5 Oviposition arena and sand intervals within each treatment ...... 40

3-6 Total number of eggs laid in each depth interval of the MCT and NT treatments...... 41

3-7 Plot of the transformed least squares means estimates for each depth interval of the MCT and NT treatments. Different letters indicate significant differences between depth intervals of the same treatment at the α= 0.05 level..... 41

3-8 Total number of eggs laid in each depth interval of the MCT and AT treatments...... 42

3-9 Plot of the transformed least squares means estimates for each depth interval of the MCT and AT treatments. Different letters indicate significant differences between depth intervals of the same treatment at the α= 0.05 level..... 42

3-10 Total number of eggs laid in each depth interval of the AT and NT treatments ..... 43

3-11 Plot of the transformed least squares means estimates for each depth interval of the AT and NT treatments. Different letters indicate significant differences between depth intervals of the same treatment at the α= 0.05 level..... 43

4-1 Egg chamber containing 30 S. abbreviatus eggs with Popsicle stick roof to hold the top sand layer...... 49

viii 4-2 The completed egg chamber (left) and the PVC tubing, 5.08 cm in diameter, used to create the egg chambers (right)...... 49

4-3 Completed apparatus to test survival of P. aequinoctialis in a PVC tube 40 cm long containing an egg chamber located 30 cm deep...... 49

4-4 Percentage of successful replications when mole cricket egg chambers were located at depths ranging from 5-30 cm...... 50

A-1 Female 1, weekly total number of eggs laid after the start of oviposition...... 54

A-2 Female 2, weekly total number of eggs laid after the start of oviposition...... 54

A-3 Female 3, weekly total number of eggs laid after the start of oviposition...... 55

A-4 Female 4, weekly total number of eggs laid after the start of oviposition...... 55

A-5 Female 5, weekly total number of eggs laid after the start of oviposition...... 56

A-6 Female 6, weekly total number of eggs laid after the start of oviposition...... 56

A-7 Female 7, weekly total number of eggs laid after the start of oviposition...... 57

A-8 Female 8, weekly total number of eggs laid after the start of oviposition...... 57

A-9 Female 9, weekly total number of eggs laid after the start of oviposition...... 58

A-10 Female 10, weekly total number of eggs laid after the start of oviposition ...... 54

ix Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

REPRODUCTIVE STRATEGY OF Pheropsophus aequinoctialis L: FECUNDITY, FERTILITY, AND OVIPOSITION BEHAVIOR; AND THE INFLUENCE OF MOLE CRICKET EGG CHAMBER DEPTH ON LARVAL SURVIVAL

Aaron Scott Weed

August 2003

Chair: J. Howard Frank Major Department: Entomology and Nematology

Scapteriscus mole crickets are serious pests of turf and pasture grasses and, to lesser extent, vegetable seedlings in the southeastern Unites States. Pheropsophus aequinoctialis is native to South America and is currently under examination because the larvae are specialist predators of mole cricket eggs. Our study examined the fecundity, fertility, and oviposition behavior of P. aequinoctialis females; and the influence of mole cricket egg chamber depth on larval survival. Results indicated that females began laying eggs 1 month after emergence; and began laying fertile eggs 2 months after emergence.

The fecundity of laboratory-reared adult beetles varied greatly between females. No relationship was observed between fecundity and the proportion of fertile eggs. In oviposition experiments, females laid most eggs near the sand surface; and preferred to lay eggs in sand with mole cricket tunnels rather than in sand with artificially created tunnels or in sand without tunnels. Physical tunnel structure was not an important cue for

x oviposition; but influenced the depth at which eggs were laid. Oviposition appeared to be influenced by a cue associated with mole cricket-excavated tunnels. Finally, mole cricket egg chamber depth did not affect larval survival. We discuss the evolution of the observed reproductive strategy for P. aequinoctialis in relation to its life history requirements.

xi CHAPTER 1 REVIEW OF LITERATURE

Mole Cricket Diversity in the Southeastern United States

Four species of mole crickets (Orthoptera: Gryllotalpidae) occur in the southeastern United States; one is native and three are exotics from South America. The exotics are considered serious pests. The native species Neocurtilla hexadactyla (Perty) is widely distributed in the southeastern US and is not considered a pest. The immigrant pest species Scapteriscus borellii Giglio-Tos (southern mole cricket), S. vicinus Scudder

(tawny mole cricket), and S. abbreviatus Scudder (shortwinged mole cricket) are important pests to turf and pasture grasses (and crop plants, to a lesser extent). Currently, the range of S. borellii and S. vicinus stretches from North Carolina to Florida and west to

Texas. Only S. borellii has been reported west of Texas from Arizona and California.

Scapteriscus abbreviatus is restricted to the points of its arrival, perhaps because it does not have the ability to fly. It occurs in coastal areas of peninsular Florida and in a few small isolated populations in inland Florida (Frank and Parkman 1999).

Mole Cricket Biology

Mole crickets spend most of their lives underground in vertical and horizontal tunnels (sometimes called galleries). They mainly burrow in the top 20 to 25 cm of soil

(Hudson 1985). The depth at which they burrow is largely a function of the soil moisture and temperature. Most activity in and outside the burrows occurs from late afternoon to around midnight (Hudson 1985; Walker 1985). Adults are usually active in the spring and autumn, but this is variable among the three Scapteriscus species. In Florida, S.

1 2 vicinus commonly is active in the spring about 3 weeks earlier than S. borellii; but both are active at about the same time in the autumn months. Both species pass the winter as large nymphs (Frank and Parkman 1999). Nymphs appear as early as April; and take around 5 months to mature to adults during the summer (Walker 1985). In contrast, S. abbreviatus has two reproductive peak periods (late spring and winter); with all developmental stages occurring throughout the year in Florida.

Each Scapteriscus species in northern Florida, and throughout the rest of their northern range, completes one life cycle every summer. However, in southern Florida

S. borellii may go through two generations in the summer (Walker 1985). A nymph requires vast amounts of food for successful development; and may molt 8 to 10 times before becoming an adult. Scapteriscus vicinus and S. abbreviatus are primarily herbivorous; whereas the diet of S. borellii is mainly carnivorous (Matheny 1981). Thus, the damage produced by S. borellii is mainly due to active burrowing and disruption of the soil surface, which can desiccate the soil and mechanically injure and uplift the plants. In contrast, S. vicinus and S. abbreviatus damage plants by directly feeding on the roots and shoots; and by burrowing behavior similar to that of S. borellii.

Mole cricket mating occurs mainly in the spring and autumn months. Males of

S. vicinus and S. borellii call for about an hour, starting 10 to 20 minutes after sundown, with loud species-specific songs to attract females. The ability of a male to attract a receptive female is due to the loudness of the song, which is determined by the size of the male and the moisture content of the soil where the chamber is constructed (Forrest

1985). Males of S. abbreviatus do not produce loud calls, but rather soft chirps for an unknown social purpose (Frank and Parkman 1999).

After copulation, Scapteriscus females burrow into the ground until an appropriate depth is reached to deposit their eggs, which is largely dependent on the soil type and moisture. Forrest (1985) found that mole crickets commonly deposited eggs 9 to 30 cm deep. However, Lake (2000) observed that S. abbreviatus deposited eggs up to a depth of

72 cm in the laboratory. From 25 to 60 eggs are laid in each chamber. After oviposition, the Scapteriscus female closes the chamber and leaves. In contrast, laboratory observations suggest that Neocurtilla hexadactyla builds an adjoining chamber to tend the eggs at least until they hatch. Eggs typically hatch within 3 weeks depending on the soil temperature.

Mole Cricket Damage

In 1986 it was estimated that, in Florida, annual costs attributed to mole cricket damage and their control in turf grass amounted to around $45 million (Frank and

Parkman 1999). In 1996, annual estimates for chemical control in Florida were greater than $18 million; and in Georgia greater than $12 million (Frank and Parkman 1999).

Significant losses occur mainly to the turf grass and cattle industries, but substantial losses can also occur to crop plants. Immigrant mole crickets are pests in the US and also in other countries such as Puerto Rico, Australia, and Cuba. In these countries the pests are not solely Scapteriscus spp. but in most cases are non-native immigrant species

(Frank and Parkman 1999). The situation in these countries is much the same as in the

US; large amounts of damage to crop plants and turf grass require spending large sums of money for control by current conventional methods.

The damage to turf and pasture grasses and crop plants occurs from voraciously feeding nymphs; and from burrowing, which dislodges or loosens the soil, desiccating the

4 plant. The common grasses planted in Florida and subject to economical loss by mole cricket damage include:

• Bahiagrass (Paspalum notatum Fluegge) • Bermudagrass (Cynodon spp.) • Centipedegrass (Eremochloa ophiuroides (Munro) Hack) • St. Augustinegrass (Stenotaphrum secundatum (Walt.) Kuntze) • Zoysiagrasses (Zoysia spp.) • Floralta limpograss (Hemarthria altissima [Poir.] Stapf and C. E. Hubb)

Typically, damage to these turf- and pasture grasses is influenced by a combination of mole cricket damage, fertilization, soil acidity, mowing height, and the intensity of livestock grazing (Adjei 2000; Frank et al. 2002).

Mole crickets also can be serious pests to a number of crop plants (Hudson 1985).

Typically, young or newly transplanted seedlings are the most susceptible. Damage usually occurs on the stems, roots, and lower leaves; but is also common on the subterranean parts of radishes and plants with tubers. Feeding damage is cutworm-like in appearance and increases the chance for establishment of plant pathogens (Schuster and

Price 1992).

Control of Pest Mole Crickets

Many control strategies have been used (with varying levels of success) against pest mole crickets. In the past, chemical control was the principal means and is still used today to protect turfgrasses. Cultural manipulation and the development of resistant grass cultivars also have been used but with little effectiveness. Currently, successful mole cricket control is achieved through an integrated pest management strategy of chemical and biological control.

The subterranean lifestyle of mole crickets and their great mobility make population estimates difficult (Hudson and Saw 1987). Damage thresholds are difficult

5 to predict because of problems with sampling procedures and problems correlating samples with the true population size (Hudson 1985). Current methods for sampling mole cricket populations include soap flushing, linear pitfall traps, and sound traps with male calling songs of S. borellii and S. vicinus. Hudson (1989) developed an equation to predict true population size from soap flush samples. He found true population estimates within 25% of the predicted value. The best estimates occurred under higher soil moistures, within ~19.5% of the predicted value. Cobb and Mack (1989) compared soap flush catches of S. vicinus to a rating system for evaluating damage; and found that they could adequately relate S. vicinus densities to the potential amount of damage. This approach did not work well for extremely high densities of mole crickets, but was a satisfactory method for estimating damage when densities of young nymphs were low.

These sampling methods are not always accurate and may delay control measures. A more recent approach that limits insecticide applications includes mapping continually infested areas and scouting for damaged turf.

Chemical Control of Mole Crickets

Chemical control from the early 1900s until the 1940s consisted of chemical baits containing calcium arsenate or calcium cyanide (Kepner 1985). These baits proved useful until newer, more effective chemicals came onto the market in the mid 1940s (in the form of DDT and chlordane). During the 1940s, chemicals produced the best results compared to all other alternatives; but after extended use, residues began to appear on crop plants and resistance even to chlordane was documented (Kepner 1985). By the

1970s, the EPA banned the use of DDT; and this forced pest managers to search for alternative methods to control mole crickets. Although new chemicals come to market

6 each year, the EPA continues to restrict many chemicals because of the potential danger of these chemicals to the environment, humans, and water sources.

Pest managers have turned to the use of juvenile hormone (JH) analogs to prevent successful development of mole cricket nymphs. Parkman and Frank (1996) found that the JH analog fenoxycarb in a bait, significantly reduced egg hatch of S. abbreviatus and produced deformities in the adults that survived the baits. However, aqueous application of fenoxycarb to the nymphs did not affect reproduction of the surviving adults. Another

JH analog, hydroprene, affected neither survival nor development of S. abbreviatus nymphs nor reproduction of the mature adults (Parkman and Frank 1998). However, they did observe significant mortality, lengthened developmental times of the nymphs, and increased susceptibility of adult females after treatments with another JH analog, pyriproxyfen.

Irrigation has long been thought to influence the efficacy of insecticides. Xia and

Brandenburg (2000) examined the affects of irrigation before and after insecticide sprays of bifenthrin and imidacloprid to determine the influence irrigation may have on insecticide efficacy against S. vicinus and S. borellii. Their results suggested that irrigation before, after, or before and after treatment did not affect the efficacy of the insecticides.

Overall, chemical control is expensive and does not provide permanent control of mole crickets. Finding the “ideal” chemical may never happen because of the possible development of resistance and continuous pressures of the EPA. Current methods that track recurrent infestation sites of Scapteriscus mole crickets and anticipate egg hatch so

7 that small nymphs can be targeted has proved beneficial to limit the number of insecticide sprays.

Cultural Control to Prevent Mole Cricket Damage

Physical disturbance to the soil to desiccate mole cricket eggs, flooding, and mowing turf to particular heights have all been tested for their effectiveness in preventing mole cricket damage but have yielded little success in the US. Production of resistant strains of grass has been suggested as a means to provide a safe and economical means of control (Reinert and Busey 1985), but resistant grasses do not affect nymphal survival of mole crickets to any great extent (Braman et al. 2000; Hudson 1986). Every tested turf and pasture grass cultivar displays variability in susceptibility to mole cricket damage

(Braman et al. 2000; Hudson 1986; Reinert and Busey 1985).

Braman et al. (2000) and Reinert and Busey (1985) observed that turf grass varieties of a finer texture were attacked more frequently and were less tolerant to damage than those with a coarser texture. Braman et al. (2000) found ‘Tifsport’ Bermuda grass and seashore paspalum most tolerant to mole cricket damage when compared with other Bermuda and paspalum grass cultivars, but neither was completely resistant.

Hudson (1986) found pasture grass Hemarthria spp. cultivars to be more tolerant to

S. vicinus damage than Bahiagrasses, but when no alternative food source was available

Hemarthria spp. cultivars still suffered severe damage.

Biological Control of Mole Crickets in Florida

Biological control has the potential to provide permanent, area-wide protection at a lower cost than alternative control methods (Frank and Parkman 1999). Three biological control organisms - a tachinid fly, a nematode, and a wasp - have been introduced from regions in South America where Scapteriscus mole crickets are native. Successful

8 establishment has occurred in Florida and, given time, the combined efforts of all three organisms should provide substantial control of the pest mole crickets and may even eliminate the use of harmful pesticides in some areas.

A tachinid fly, Ormia depleta (Wiedemann), was released in Florida in 1988 and was established throughout 38 Florida peninsular counties by 1994. Populations appeared to be restricted to the southern parts of Florida because the adult flies could not survive during the winter months in northern Florida (Frank et al. 1996; Walker et al.

1996). In an attempt to expand the northern range of this fly, a strain of O. depleta collected from a temperate area of Brazil was released into Florida and other states such as Louisiana, North Carolina and South Carolina in the years 1999-2002. No indication of establishment in these areas after these releases has been reported.

Counties reporting annual populations of O. depleta have recorded significantly less mole cricket damage than those which have yet to be colonized (Frank et al. 1996).

In their review of mole cricket pest management, Frank and Parkman (1999) claim nectar sources for the adult flies need to be planted in areas containing mole crickets in order to support and increase O. depleta populations. However, a recent examination of

O. depleta crop contents discovered these flies are consuming primarily honeydew rather than nectar (Welch 2000). Therefore, encouraging plants that support honeydew- producing insects in mole cricket infested areas may sustain local fly populations and increase parasitism levels.

An entomopathogenic nematode, Steinernema scapterisci Nguyen & Smart, was released in Florida pastures during 1985 and has since established populations with the potential to spread (Frank and Parkman 1999). The nematode successfully attacks adults

9 and large nymphs of S. borellii and S. vicinus. Drawbacks of the nematode include its ineffectiveness against S. abbreviatus and small to moderately sized Scapteriscus mole cricket nymphs (Parkman and Frank 1993). The nematode also needs continuously moist soils to spread and survive. In 1990, S. borellii and S. vicinus were inoculated with

S. scapterisci by attracting the mole crickets to sound traps baited with infective juvenile nematodes on 21 golf courses. Of the 21 golf courses, seven later contained nematode- infected mole crickets (Parkman and Frank 1993), and it is evident that they can maintain constant populations in Florida. Currently, S. scapterisci is manufactured as a biopesticide and also acts as a classical biological control organism.

Larvae of the digger wasp Larra bicolor Fabricius are ectoparasitoids of large

Scapteriscus nymphs. Fortunately, the adults will not attack the native mole cricket

N. hexadactyla. Larra bicolor was initially released into Florida in 1981 from a Puerto

Rican stock that failed to spread and produced little effect against mole cricket populations (Castner 1988; Frank and Parkman 1999). Later, a slightly more cold- tolerant biotype from Bolivia was introduced and it seems to survive the winters in

Florida because it is spreading (Frank and Parkman 1999). Recently, J. H. Frank (pers. com.) estimated that the parasitoid achieved ~70% reduction in mole cricket populations in north central Florida. Similar to O. depleta, planting the preferred food source, in this case nectar, for the adult wasps should help spread and increase the parasitoid’s effectiveness at controlling pest mole crickets. Southern larraflower, Spermacoce verticillata L., has been noted as one of a few preferred plants of L. bicolor and is currently being planted in various parts of Florida in order to monitor and help spread the wasp. However, it should be noted that J. H. Frank (pers. com.) observed that when

S. verticillata flowers were not present in their plots the same parasitism levels were reached suggesting that L. bicolor will use alternate nectar sources.

Other biological agents considered have been the fungal pathogens Metarhizium anisopliae and Beauveria bassiana. Metarhizium anisopliae has potential to reduce mole cricket populations but its virulence is dependent on the strain and on the species of mole cricket. Furthermore, to be effective it needs to be able to produce progeny conidia in a field setting (Boucias 1985). Beauveria bassiana was recently observed producing levels of control similar to those of the insecticides imidacloprid, bifenthrin, and deltamethrin

(Xia et al. 2000). It is not likely these fungal pathogens could be released into the environment and be expected to sustain populations as would a classical biological control agent because of their high susceptibility to environmental conditions. The use of these organisms as biopesticides applied in an augmentative fashion seems more likely and may provide adequate control of small nymphs and S. abbreviatus mole crickets

(Frank and Parkman 1999) when applied at high concentrations and under the correct environmental conditions.

South American bombardier beetle Pheropsophus aequinoctialis L. (Coleoptera:

Carabidae: Brachinini) could be considered as another biological control agent of

Scapteriscus because the larvae may be specialist predators of Scapteriscus eggs. The beetle has not been released in the US due to minimal knowledge of its behavior and the possible non-target affects that may occur in the field. In addition, more conclusive evidence is needed to describe its potential to reduce pest mole cricket populations that may be established in riparian areas that cannot be subjected to chemical applications

(Frank and Parkman 1999). The next section provides background information on

11 ground beetle ecology and focuses on examples of other ground beetles with a similar lifestyle to that of P. aequinoctialis.

Carabid Beetle Ecology

Erwin (1991) estimated that approximately 40,000 species of Carabidae have been described worldwide. Their highly adapted structure and life cycles have enabled members of the Carabidae to inhabit virtually all terrestrial habitats since the Mesozoic.

The group is abundant worldwide, but the vast amount of literature is mainly biased towards the temperate zones of the Northern Hemisphere (Lövei and Sunderland 1996).

Temperate carabid beetles typically are univoltine, but bi- and multivoltine life cycles do exist elsewhere (Lövei and Sunderland 1996). There are usually 3 instars (see

Erwin and Erwin (1976) and Lövei and Sunderland (1996) for exceptions) and the second or third instar undergoes diapause in the temperate zone. The larvae are typically free- moving polyphagous carnivores that feed on extraorally digested food and pupate in specially constructed cells in the soil or under bark.

The adults can be long lived and diurnal species typically hunt by vision while nocturnal species usually use olfactory-tactile cues. Specifically, some adults are reported to detect prey by chemical cues given off from aphids (Chiverton 1988), springtails (de Ruiter et al. 1989), and snails (Wheater 1989). It should be noted that not all carabids are predatory, in fact, most appear to be scavengers on recently injured or killed insects. Many carabids also are known to be omnivorous while others are strictly herbivorous (Lindroth 1949). Adult carabids typically find suitable habitats based on abiotic cues, internal clocks, and presence of appropriate food (Thiele 1979). Evans

(1988) demonstrated that some riparian adult ground beetles use the volatile chemicals emitted from blue algae to locate a suitable habitat.

Most females lay eggs singly (Lövei and Sunderland 1996), while those that lay eggs in large batches do so in prepared oviposition sites (Thiele 1977). Fecundity varies greatly between species (Thiele 1977) and is determined largely by composition of the adult’s prey (Wallin et al. 1992) and by adult size, which Juliano (1985) observed with

Brachinus lateralis Dejean females. In addition, Nelemans (1987) demonstrated that the larval feeding condition largely influenced the size, fecundity, and fertility of emerging

Nebria brevicollis (F.) adults.

Pheropsophus aequinoctialis differs from most carabid beetles because its larval stage is thought to develop only on mole cricket eggs. Erwin (1979) estimated that 24% of ground beetles have life histories closely associated with other invertebrates. These unique associations have been reported in 7 tribes and hypothesized to exist in 11 others

(Erwin 1979). Ant symbionts are found in 3 tribes, termite symbionts in 1, and the other

3 tribes include individuals with specialist ectoparasitoid or predatory behavior (Erwin

1979). P. aequinoctialis belongs in the latter group. However, not every member of these tribes exhibits these traits. Carabid inquilines in ant and termite nests typically mimic the semiochemicals within these social insect communities to their advantage, but these carabids will not be the focus of this review.

Understanding the intricate interactions that exist between specialist ectoparasitoid or predatory carabids and their host is often a difficult task because knowledge of the host also is needed. Determining whether an adult ground beetle eats exclusively one prey item is difficult and studies concerned with this issue provide only assumptions based on close ecological associations. Such assumptions exist in studies describing the association of ground beetles with snails (Lindroth 1949, pg 486), millipedes (MacSwain

13 and Garner 1956), and earthworms (Löser 1972). Better understanding exists concerning the associations of larval carabids with their hosts. Acknowledged examples of carabid- host associations occur in the tribes Peleciini, Lebiini, and Brachinini, with the latter two more thoroughly evaluated. Based on reported descriptions of the brachinines, some general conclusions about these carabids are as follows: (1) the 1st instar typically is reduced in size and develops only on one host successfully (Liebherr and Ball 1990), (2) once feeding has started the larva goes through hypermetamorphosis, whereby it loses its ease of movement and increases in volume dramatically so that the late instar is confined until the adult emerges, and finally (3) the size of the larval host has a major influence on the size of the newly emerging adults (Erwin 1967; Juliano 1985). The following sections will summarize individual life histories of certain members from each of these 3 tribes.

Tribe Peleciini

Members of this tribe are not well understood and the only published life history account is from field observations completed by Salt (1928) in Colombia on Pelecium sulcatum Guérin. The author collected 6 larvae in the field, one being attached to an unidentified beetle pupa, one attached to a chrysomelid pupa, and the four remaining were discovered in millipede chambers 4 to 8 cm beneath the soil. An adult was only obtained from one larva eating the young millipedes and this was assumed to be the preferred host by the author, but Erwin (1979) assumed this species to also be associated with chrysomelid pupae. It was hypothesized that females laid eggs on the host or that the small larva is the host seeking stage (Salt 1928).

Liebherr and Ball (1990) investigated characters of first instars of Eripus oaxacanus Straneo & Ball and determined they were consistent with the derived

14 characters of other larval carabids with a parasitic lifestyle (e.g., labium lacking a ligula).

Although no natural history reports have been made, the authors determined that the larvae appeared to be specialist predators because they refused mealworm larvae, which most larval carabids will consume.

Tribe Lebiini

One of the first complete accounts recognizing a carabid with this specialized lifestyle was completed by Silvestri (1905), who described the association of Lebia scapularis Fourcroy with the elm leaf beetle Galerucella luteola Müller in southern

Europe. He reported the 1st instar as having long legs in order to seek its host and large mandibles to penetrate the host’s cuticle. After feeding on the host pupa began, the larva soon lost its ability to move easily because the body widened and the legs shortened.

Finally, the host was consumed and the larva molted into a pupa in the elm leaf beetle’s pupal chamber.

Erwin and Erwin (1976) described the life history of a South American ground beetle, Eurycoleus macularis Chevrolat, and its association with a fungus inhabiting

Amphix spp. (Coleoptera: Endomychidae). After adult eclosion, male and female

E. macularis moved to patches of polypore fungus where they began to mate. Eurycoleus macularis females then dispersed to logs with Amphix colonies and deposited an egg clutch within centimeters of a large number of Amphix pupae. The authors assumed the females were chemically orienting to the Amphix colonies. When the eggs hatched, the larvae attached themselves to Amphix pupae and consumed the host. Pupation occurred after the 4th instar under the bark in constructed pupal chambers.

Other reports concerning lebiines and their hosts include a brief description of the association of Lebia chlorocephala Hoffm. with Chrysomela spp. especially Chrysomela

15 varians Schall (Lindroth 1954) and the association of Lebia grandis Hentz with the

Colorado potato beetle Leptinotarsa decemlineata (Say) in North America (Chaboussou

1939).

Tribe Brachinini

North America members of this tribe include species in the genus Brachinus, which have all been reported as being specialized ectoparasitoids of Dytiscidae (Juliano

1984), Hydrophilidae (Erwin 1967), and Gyrinidae (Dimmock and Knab 1904; Wickman

1894) pupae. The adult beetles are small to medium sized carabids that inhabit the shores of streams, lakes, and ponds (Erwin 1967). Of the 62 species of Brachinus represented in

North and Middle America (Erwin 1970), only one paper written by Erwin (1967) has given an in depth description of natural history of one species. The author observed

Brachinus pallidus Erwin adults mating at night and into the morning hours as early as

May and lasting until September at Coyote Creek, Santa Clara County, California. In the morning at the stream’s edge females inserted an egg into a previously prepared mud ball and deposited the mud ball on a rock. After the egg hatched, the 1st instar actively searched for a host. After penetration into a hydrophilid pupal chamber, the larva attached itself to the pupa and began feeding. After about an hour, the larva molted into a very different looking 2nd instar. In this larval stage the mandibles changed in appearance and the legs were short and thickened. As feeding continued, the body progressively thickened and after the host was completely consumed a 5th instar was reached. Pupation took place within the host’s pupal chamber and the adult emerged in about 8 days at room temperature.

Habu and Sadanaga (1965) recognized the association of a brachinine carabid with a mole cricket (Orthoptera: Gryllotalpidae) in Japan. In their paper they provide larval

16 descriptions and natural history notes on Stenaptinus jessoensis (Morawitz). From their observations they found that the adults were polyphagous predators, but larvae only developed on eggs of Gryllotalpa africana Palisot. However, because G. africana is now believed to occur only in Africa (Townsend 1983), it is likely the eggs were of another

Gryllotalpa species. Adult beetles were very widespread in Japan and typically found in paddy fields. In the laboratory they determined that the females were very fecund, laying a large number of eggs in masses. Oviposition occurred from mid-June until late July.

They also determined that there were 3 larval stages, all being noticeably different from one another. The 1st instar was very active and had the ability to survive for long periods without food. As the larva progressed from 1st to 3rd instar, the legs became shorter and the body widened remarkably. The pupal stage was only mentioned briefly. Newly emerged adult beetles would dig their way to the soil surface out of the mole cricket egg chambers.

Currently, the Entomology and Nematology Department at the University of

Florida maintains a colony of Pheropsophus aequinoctialis L. that originated from

Bolivia in association with pest Scapteriscus mole crickets. Like Stenaptinus jessoensis,

P. aequinoctialis larvae appear to develop only on mole cricket eggs and the adults are also polyphagous predators. Frank and Hemenway determined in quarantine studies that

P. aequinoctialis larvae developed on eggs from the three Scapteriscus spp. in Florida and N. hexadactyla mole crickets (unpublished data). They also determined that at 27°C, on average, each developmental stage of P. aequinoctialis took the following number of days to develop: egg, 14; 1st instar, 7; 2nd instar, 4; 3rd instar, 15; and pupa, 20. In addition, Frank and Hemenway (unpublished) also determined that first instars could

17 survive up to 11 days on average without food and about 30 eggs were needed for the larvae to develop into adults.

Other studies concerning P. aequinoctialis include observations on behavior of the adults and larvae and a census of the invertebrates the larvae may encounter at lakeshores in Florida (Bertorelli 1998; Lake 2000). Lake (2000) demonstrated that female adult beetles were not attracted to Scapteriscus abbreviatus eggs in a Y-tube olfactometer and

1st instars did not rely on the mole crickets to transport them to the egg chamber. Lake

(2000) found that 90% of the beetle eggs were deposited 0 to 15 cm from the soil surface.

In addition, 1st instars placed directly on egg clutches in the lab did not eat the eggs and therefore did not develop successfully, while those allowed to burrow to the egg chamber readily ate the eggs and developed successfully (Lake 2000). From these observations it was assumed the 1st instars use the act of burrowing to initiate feeding behavior.

Bertorelli (1998) inventoried lakeshores in Gainesville and determined that due to the small size and aquatic nature of the predominant invertebrates collected, they did not possess likely characteristics to be considered as potential prey for P. aequinoctialis larvae. However, further evidence is still needed to conclude whether any of the invertebrates inventoried could potentially be a larval prey item.

Objectives

The release of P. aequinoctialis into Florida for control of Scapteriscus mole crickets has been delayed due to incomplete knowledge concerning the biology, potential non-target affects, and its ability to control mole crickets. Lake (2000) investigated the oviposition behavior of P. aequinoctialis, but did not give the beetles an opportunity to move both horizontally and vertically, which may have influenced the results. In addition, beetles were not given choice tests to oviposit in, which could have allowed him

18 to determine whether the beetles randomly deposited eggs over the soil surface or, for instance, concentrated oviposition in mole cricket tunnels. In other words, no attempt was made to determine whether female beetles would oviposit differently when mole crickets were present, something worth investigating with a carabid of this nature.

Furthermore, Lake (2000) stated that the depth and placement at which adult females lay their eggs might influence larval survival because the larvae need to burrow to the egg chambers. Again, an arena with choice tests may have helped to determine this. The objectives of this study are to supplement the aforementioned study and focus on determining:

• the fecundity and fertility of females, • whether oviposition is influenced by the presence of mole cricket tunnels, • whether oviposition is stimulus-directed, and • whether the depth of the mole-cricket egg clutch influences larval survival.

CHAPTER 2 FECUNDITY AND FERTILITY OF LABORATORY-REARED Pheropsophus aequinoctialis

Introduction

Fecundity and fertility of ground beetles vary greatly among species (Thiele 1977), possibly because of the large number of species each with different life history requirements distributed over a large number of habitats. Many fecundity studies concerning carabids have typically considered the influence of diet (Ernsting et al. 1992;

Wallin et al. 1992; Juliano 1986; Spieles and Horn 1998), adult age (van Dijk 1972;

Gergely and Lövei 1987), and adult size (Juliano 1985) on the production and size of eggs.

Collecting individuals from the field and counting the number of eggs in the ovaries has been one approach for studying ground beetle fecundity. However, because

Pheropsophus aequinoctialis L. is still in quarantine facilities, only laboratory studies were permissible and, to preserve colony size, few adults could be killed. The objective of this study was to determine the fecundity and fertility over a 20-week period of 10 newly emerged P. aequinoctialis females raised on the same diet. Given that in the literature only contradictory evidence exists describing the influence of age on egg production of ground beetles this was a question worth investigating for

P. aequinoctialis. In addition, this information may be critical when releasing

P. aequinoctialis adults into Florida so fecund females can be released to increase the

19 20 chances of establishment and allow the beetle to act as a classical biological control agent against Scapteriscus mole crickets.

Materials and Methods

Experimental Colony

Ten newly emerged female and 20 newly emerged male Pheropsophus aequinoctialis adults were obtained from normal rearing procedures (Lake 2000) to monitor fecundity. Adults used in the experiment emerged over a 2 month period

(Table 2-1). Each female and 2 males were placed into a 236.6 ml (8 fl oz) plastic delicatessen cup with a moist paper towel on which to oviposit. The research colony was held in clear plastic boxes measuring 24.5 ∗ 30.5 ∗ 10 cm and placed in a Florida Reach-

In unit (Walker et al. 1993) kept at 27ºC, 10:14 (L:D) h, and 38-54% relative humidity.

Paper towels were kept moist by misting with a water bottle daily. After beetles emerged they were fed only from Monday until Wednesday of every week to keep consistent with the normal rearing procedures on a diet of Tenebrio molitor L. pupae (Coleoptera:

Tenebrionidae), oats, and raisins. The cups and paper towels were replaced after feeding on Wednesday.

Egg Collection and Fertility Determination

Eggs were collected from each cup with a fine camel hair brush every Monday and

Wednesday for 20 weeks after each female began laying eggs. All eggs were counted and placed into 5 cm petri dishes lined with a moist paper towel disk. Eggs were checked for fertility 3 times over a period of 2 to 3 weeks and the number of fertile eggs laid by each female for each week was recorded. Diseased eggs were removed when discovered and counted as infertile. Fertile eggs were removed and placed into a separate 5-cm petri dish so the emerging larvae would not cannibalize one another and possibly generate an

21 incorrect count. Weekly totals were calculated by combining the egg and fertility counts of every Monday and Wednesday of the same week. The mean number of eggs laid per week per female with 95% confidence intervals and the proportion of fertile eggs laid by each female over the 20-week period were computed. A linear correlation between fecundity and the proportion of fertile eggs for all females over the 20-week period was tested with SAS PROC GLM (SAS Institute Inc. 2000). To determine whether beetle age influenced the production of eggs, weekly egg counts over all females were transformed to the power of 0.3 to fit a linear model and analyzed as a repeated measures experiment using SAS PROC MIXED (SAS Institute Inc. 2000). The random variables included in the analysis were female and week, and the repeated measures experimental design was chosen because eggs were collected over a period of time. Means were separated by a least squares means procedure and statistical significance was determined at the α= 0.05 level. Marinela Capanu of the Department of Statistics, Institute of Food and

Agricultural Sciences, Statistical Consulting Unit, University of Florida provided statistical guidance.

Results

Females took an average of 31.6 days to lay eggs and took an average of 58.13 days to lay fertile eggs after emergence. Graphs displaying the total number of eggs laid per week for each female over the 20-week period are in the Appendix. The weekly mean number of eggs laid by each female ranged from 5.55 to 83.2 (Figure 2-1).

There was not a significant linear relationship between fecundity and the proportion of fertile eggs laid over the 20-week period for all females (F= 3.66; df= 1,8; P= 0.0922).

Females 5 and 7 laid the fewest number of eggs out of all females and never laid a fertile

22 egg over the 20-week period (Figure 2-2). Females 3 and 6 laid the highest proportion of fertile eggs, with respective proportions equaling 0.27 and 0.30 (Figure 2-2).

The weekly mean number of eggs laid by all females generally increased each week throughout the 20-week period (Figure 2-3). The week had a significant effect on the number of eggs laid (F= 3.04; df = 19,171; P<0.0001). The mean numbers of eggs laid per week during the first 4 weeks for all females were significantly different from one another and from all remaining weeks. Week 5 was significantly different from

Weeks 7, 8, 10, 11, 16, 18, and 19. After Week 5, beetles laid a similar number of eggs with the exception of Weeks 15 and 16, which were significantly different from each other (P= 0.0463).

Discussion

In the laboratory, Pheropsophus aequinoctialis females exhibited a high degree of variation in fecundity and low fertility when fed a diet of Tenebrio pupae, oats, and raisins. The reasons for this variability in fecundity between females remain unknown from these data. Perhaps a positive correlation exists between body size and the number of eggs similar to the observations by Juliano (1985). However, this could not be tested in this experiment because females were discarded before elytron length and weight measurements were taken. These results appear to be consistent with observations made by Thiele (1977), who stated that the number of eggs laid by laboratory-reared ground beetles of the same species tend to vary. Adult food consumption is also recognized as a major variable influencing fecundity (Lövei and Sunderland 1996) and may also have contributed to the outcome of my results. However, there was no indication of how much food each female beetle consumed so the influence of diet remains unknown.

There was not a significant linear relationship between female fecundity and the proportion of fertile eggs laid. However, because the regression was approaching significance, a relationship might have been observed if more females were considered in this experiment. The low fertility level of females may be the result of several factors.

Perhaps the male’s sperm was not viable or mating did not take place. Mating success of

Brachinus lateralis Dejean males was positively correlated with adult size (Juliano 1985).

Therefore, male size may have had some influence on the mating behavior of

P. aequinoctialis. However, R. Hemenway (personal communication) observed that when wild P. aequinoctialis were initially imported from South America, the beetles achieved a very similar level of fertility.

Females began to lay eggs about a month after emergence and took on average 2 months to begin laying fertile eggs. For the most part, after 2 months (or 4th week of egg collection) the beetles started to increase their overall egg production for the entire experimental period. After the 5th week, females laid a fairly constant weekly number of eggs with the exception of week 15. Females that laid the highest mean number of eggs per week produced a high number of eggs earlier in the 20-week period. Those females that laid a relatively smaller weekly mean number of eggs tended to lay the majority of their eggs towards the end of the experimental period.

Over the 20-week period, Females 5 and 7 never laid a fertile egg. After the

20-week period Female 7 was coupled with males initially in the same cup as female 8.

After 2 weeks of egg collection, Female 7 laid 9 fertile eggs out of a total of 69, or 0.13.

It appears that either the male’s sperm or mating behavior influenced the ability of

Female 7 to lay a fertile egg during the 20-week period. The same approach was used

24 with Female 5, but males previously in the cup with Female 3 were coupled with Female

5 in this trial. In total, 9 eggs were laid over a 2-week period and zero eggs were found to be fertile. Either this female did not have the ability to lay fertile eggs or the males were not mating with her.

Lake (2000) found the bacterium Wolbachia in the P. aequinoctialis colony.

Although the colony was heat-treated for Wolbachia shortly after discovery, there is no indication that the bacterium was eliminated. Therefore, Wolbachia may have had some effect on the reproduction of P. aequinoctialis. It is known to influence insect reproduction. In addition, mating was expected to occur continuously throughout the entire 20-week period. If for some reason mating by the beetles did not occur as expected, perhaps the observed fertility did not correctly reflect the actual ability of these females to lay fertile eggs. Further studies should determine the relationship of body size to egg production and to the mating success of males. In addition, increasing the length of the observation period may capture a peak egg-laying period of P. aequinoctialis females.

Table 2-1. Emergence dates (2002) of the female and 2 males placed in each of the 10 cups from which eggs were collected Cup 1 2 3 4 5 6 7 8 9 10

Female 6-May 29-May 29-May 29-May 29-May 4-June 12-June 17-June 24-June 3-July Male 1 6-May 29-May 29-May 31-May 29-May 29-May 29-May 30-June 13-June 3-July Male 2 6-May 29-May 29-May 29-May 29-May 29-May 12-June 29-June 24-June 3-July

0.40

0.30 y = 0.0001x + 0.0633 R 2 = 0.3146

0.20 Proportion of fertile eggs

0.10

0.00 0 250 500 750 1000 1250 1500 1750 Total no. eggs Figure 2-1. Mean number of eggs laid per week with 95% confidence intervals for each female

0.40

0.30 y = 0.0001x + 0.0633 R 2 = 0.3146

0.20 Proportion of fertile eggs

0.10

0.00 0 250 500 750 1000 1250 1500 1750 Total no. eggs Figure 2-2. Fecundity versus the proportion of fertile eggs laid for each female over the 20-week period

40 Mean no. eggs no. Mean

0 1 2 3 4 5 6 7 8 9 1011121314151617181920 Week Figure 2-3. Mean number of eggs laid per week with 95% confidence intervals for all females

CHAPTER 3 OVIPOSITION BEHAVIOR OF Pheropsophus aequinoctialis L. IN RESPONSE TO SAND WITH Scapteriscus abbreviatus SCUDDER TUNNELS, ARTIFICIALLY CREATED TUNNELS, AND TO SAND WITHOUT TUNNELS: DIRECTED OR RANDOM?

Introduction

Ground beetles with a larval stage exhibiting specialized predatory behavior must have a life cycle coinciding with that of its host. Host detection behavior directed either by a kairomone or another cue may have developed in response to this specialized behavior. As mentioned in Chapter 1, Erwin and Erwin (1976) studied the association of

Eurycoleus macularis Chevrolat with the polypore-fungus-inhabiting Amphix spp.

(Coleoptera: Endomychidae). Although it was not determined experimentally, the authors assumed that E. macularis adults were ovipositing near the pupae or the fungus in response to chemical cues. The authors explained that chemical receptors were fine- tuned over evolutionary time to increase the probability of finding the Amphix and fungal colonies. Other observations on the oviposition behavior of Brachinus spp. determined that females laid eggs at the water’s edge, which is the area where the water beetle pupal hosts are located (Erwin 1967 and Juliano 1985). E. macularis adults appeared to orient towards logs containing large fungal colonies or to the aggregations of the Amphix pupae, while Brachinus spp. appeared to orient towards the habitat rather than to their concealed host.

The larvae of Pheropsophus aequinoctialis are specialized predators of

Scapteriscus mole cricket eggs in South America and female beetles may have evolved

28 29 directed oviposition behavior similar to E. macularis and Brachinus spp. Mole crickets create horizontal and vertical tunnels in the soil and deposit their eggs 9-30 cm deep in underground chambers off from these tunnels (Forrest 1985). Mole cricket tunnels may signify to the female beetles that mole cricket eggs are in the area and beetles may lay their eggs in or around these tunnels. The objective of these experiments was to determine how the presence or absence of tunnels, either created by the mole cricket or artificially created, influences beetle oviposition. My goal was to determine whether female beetles are directing oviposition towards mole cricket tunnels, which may influence larval survival.

Materials and Methods

General Plexiglas Sandwich

Ovipositional substrates were constructed in sand held within Plexiglas sandwiches. The sandwiches were made out of two 30 ∗ 30 ∗ 0.5 cm Plexiglas sheets.

On one sheet, 3 Plexiglas strips, each measuring 1 ∗ 29.5 ∗ 0.5 cm, were hot glued around the edges of the sheet to separate the sheets by 1 cm when they were held together with 5 cm clips to form the sandwich. The total volume within the sandwich measured 29.5 ∗

29 ∗ 1 cm or 855.5 cm3.

To create the oviposition arena, a volume of 696 cm3 (24 ∗ 29 ∗ 1 cm) of moist, autoclaved builder’s sand was added into each sandwich so the final height of sand equaled 24 cm. This height of sand reduced the chances of the beetle escaping and allowed the sand to be horizontally partitioned into six, 4-cm intervals to determine the depth at which eggs were deposited by every female. Black construction paper, measuring 24.5 ∗ 30 cm, was attached to the outside of the sandwich and held at the sand

30 surface by the clips. The paper was used to darken the oviposition arena, simulating being underground when the mole cricket or beetle burrowed into the sand (Figure 3-1).

The sandwiches were held vertically and placed so that the broad side of the sandwich was facing the light source.

Mole Cricket Tunnel (MCT) versus No Tunnel (NT)

An oviposition arena was created with mole cricket tunnels (MCT) and with no tunnels (NT) to determine how the presence of MCT influenced beetle oviposition. After the sand was added into the sandwich, a 1 ∗ 29.5 ∗ 0.5-cm strip of sand was removed from the middle of the sand splitting the arena in half vertically to create two treatments.

A Plexiglas barrier was placed into the open area to confine the mole cricket to one half of the arena to create the MCT treatment. The sandwich sheets were closed with black construction paper on the outside, and a female S. abbreviatus was added to one half of the sandwich and left to burrow for 4 days. The sand displaced by the mole cricket was removed throughout the 4-day period so the sand depth was kept at 24 cm. After the 4th day, the sandwich was opened and the mole cricket was removed carefully and detailed drawings containing various measurements were taken of all tunnels (Figure 3-2). The barrier was removed and replaced with moist, autoclaved sand to complete the oviposition arena. The sandwich was closed with black construction paper in place, oriented vertically, and ready for the female beetle.

Mole Cricket Tunnel (MCT) versus Artificial Tunnel (AT)

An oviposition arena with MCT and artificial tunnel (AT) treatments was created to determine whether oviposition is directed towards mole cricket tunnels and how tunnel structure influences oviposition behavior. The AT treatment was created to simulate the structure of a mole cricket tunnel, but devoid of the chemical cues which might be left by

31 mole crickets in their tunnels. The dimensions of the artificial tunnel were created from computing the average total depth, average number of turns, and average starting depth of the 10 MCT’s created in the MCT versus NT arena described above. The AT was 1 cm wide throughout its length, started 1 cm down from the top of the sand, contained two turns, and ended at a depth of 22 cm. A cardboard tunnel template was created from these measurements and the tunnel was created in the sand by placing the top left-hand corner of the template 1 cm below the sand surface and 3 cm from the left-hand side of the sand edge. The cardboard template was outlined with a sterilized knife and the sand was removed with a metal spatula. The placement of the AT was kept constant between replications (Figure 3-3).

The MCT treatment was created by the same procedure as described in the MCT versus NT arena. After the MCT treatment was created, the AT treatment was made and the oviposition arena was complete with both treatments (Figure 3-4). The sandwich was closed with black construction paper in place, oriented vertically, and ready for the female beetle.

Artificial Tunnel (AT) versus No Tunnel (NT)

To determine whether tunneled sand, devoid of possible mole cricket chemical cues, provides a better ovipositional substrate than untunneled sand, an oviposition arena was created with AT and NT treatments (Figure 3-3). The AT treatment was created by the same procedure as previously mentioned in the MCT versus AT arena. After the AT treatment was created, the sandwich was closed with black construction paper in place, oriented vertically, and ready for the female beetle.

Oviposition

All female beetles used in this experiment were no more than 1 year old and presumably gravid because females are continuously kept with males in 236.6-mL (8 oz.) plastic delicatessen cups at all times. Females were randomly selected from the research colonies. Each female was placed into the middle of a vertically oriented Plexiglas sandwich and the direction they faced was alternated between replications. The top of the sandwich was covered with another Plexiglas strip to prevent the beetle from escaping and to hold in moisture. Beetles were left to oviposit for 4 days and were carefully removed with forceps after the oviposition period. Each sandwich was held in a

Florida Reach-In unit (Walker et al. 1993) kept at 27ºC, 10:14 (L:D) h, and 38-54% relative humidity over the 4-day period.

Sampling Sandwiches with Treatments

After the beetle was removed from each arena, the sand was split in half vertically with a knife to separate the treatments. This left each treatment with a volume of 348 cm3 of sand. The entire arena of sand was horizontally partitioned into six, 4-cm intervals. Twelve individual samples were obtained in each arena, with 6-depth intervals for each treatment (Figure 3-5). Each sand-interval was scraped into a 236.6-mL (8 oz.)- plastic delicatessen cup that was labeled with the appropriate depth interval and respective treatment information.

Egg Extraction

Eggs were extracted from sand by a method developed by Jenkins (1964) to extract nematodes from soil. This method was later modified by Lake (2000) to extract

P. aequinoctialis eggs from sand, which extracted eggs with 98% efficiency. Each individual sand sample was placed into a 354.9 mL (12 oz)-plastic cup. Into each cup

33 was added a 100 mL portion of a sugar solution, created by combining 300 g of table sugar to 1 L of tap water. The contents were stirred continuously until the sand was suspended in the solution and the mixture was left to settle for 5 min. The settled solution was poured off into a 9 cm petri dish and eggs were removed into water using a dropper. To make sure all of the eggs were extracted, the sand in the cup was washed a second time with water and this mixture was poured into the 9-cm petri dish. Eggs were removed from the water, counted, and placed into 5 cm petri dishes lined with moist paper towel disks to determine fertility for laboratory rearing procedures.

Ten females were individually placed into each oviposition arena for each of the three choice arenas. The mean number of eggs was computed for each depth interval within each treatment. Marinela Capanu of the Department of Statistics, Institute of Food and Agricultural Sciences, Statistical Consulting Unit, University of Florida provided statistical guidance. The data were transformed as necessary to fit the normality assumption. Data obtained from the MCT versus NT arena were transformed to a power of 0.15. The data obtained from the MCT versus AT experiment were transformed to a power of 0.2. The data obtained from the AT versus NT experiment were transformed using a logarithmic transformation. A split plot analysis was performed on all three experiments with the SAS PROC MIXED procedure (SAS Institute Inc. 2000) with the random effects being replication, and replication ∗ treatment and the fixed effects being treatment, depth, and treatment ∗ depth. In the analysis of AT versus NT, the number of eggs counted from the 0-4 cm interval was compared to all of the eggs found in the 4 to

24 cm depth intervals. This was done because out of a total of 120 observations, 88 were zeroes. In the non-zero observations, over half were in the 0-4 cm depth interval, so it

34 was appropriate to compare this interval to the 4 to 24 cm intervals. In all experiments, least squares means procedures were performed for mean separation of the main effects.

In the MCT versus NT experiment, a contrast procedure was also used for mean comparison. Statistical significance was determined at the α= 0.05 level.

Results

MCT versus NT

Of the total number of eggs laid in the oviposition arena, 33.15% were in the top

0-4 cm depth interval (Figure 3-6). Significantly more eggs were laid in the MCT treatment (836 eggs or 78%) compared to the NT treatment (238 eggs or 22%) (F=13.83; df= 1, 9; P= 0.0048). In the MCT treatment, 38.6% of the eggs were laid in the top 8 cm of sand and in the NT treatment, 93% of the eggs were laid in the top 8 cm of sand

(Figure 3-6). The depth had a significant effect on the number of eggs laid (F= 10.12; df= 5, 90; P< 0.0001).

The treatment ∗ depth interaction was not significant (F =1.24; df= 5, 90;

P= 0.2992). The results of the contrast analysis determined that the mean number of eggs laid in the 0-4-cm depth interval averaged over both treatments was significantly higher than all other intervals (t= -2.20; df= 5, 90; P= 0.0301). No significant differences were reported between any other depth intervals (Table 3-1).

The least squares means analysis reported a similar mean number of eggs in the

0-4-cm and 4-8-cm depth intervals of the MCT treatment, but more eggs in 0-4-cm than in the 8 to 24 cm depth intervals of this treatment (Figure 3-7). In the NT treatment, more eggs were laid in 0-4-cm than in the 4 to 24 cm depth intervals (Figure 3-7). In

35 addition, more eggs were laid in 4-8-cm than in the 8 to 24 cm depth intervals of the NT treatment (Figure 3-7).

MCT versus AT

About 47% of all eggs laid in the oviposition arena were laid in the top 4-cm of sand (Figure 3-8). A higher number of eggs was laid in the MCT treatment (663 eggs or

77.5%) than in the AT treatment (193 eggs or 22.5%) (F= 11.98; df= 1, 9; P= 0.0071).

The depth had a significant effect on the number of eggs laid (F= 9.50; df= 5, 90;

P< 0.0001).

The treatment ∗ depth interaction was not significant (F= 1.69; df= 5,90;

P= 0.1461). The results from the least squares means analysis indicated that in the MCT treatment, more eggs were laid in 0-4-cm than in 4 to 20 cm depth intervals, but a similar number was laid in the 0-4-cm and the 20-24 cm depth intervals (Figure 3-9). More eggs were laid in 20-24-cm than in the 4 to 12 cm depth intervals of the MCT treatment, but a similar mean number was recorded in the 12 to 20 cm depth intervals (Figure 3-9).

Similar numbers of eggs were laid in the 4 to 20 cm depth intervals of the MCT treatment

(Figure 3-9).

In the AT treatment, more eggs were laid in the 0-4-cm depth interval than in the 4 to 24 cm depth intervals (Figure 3-9). A similar mean number of eggs was laid within the

4 to 24 cm depth intervals.

AT versus NT

Sixty six percent of eggs laid in the oviposition arena were found in the 0-4 cm interval (Figure 3-10). About 56% of the eggs were laid in the AT treatment, and within this treatment, almost 50% were laid in the 0-4-cm interval of sand (Figure 3-10). In the

NT treatment, eggs were only extracted from the top 8 cm of sand, with 86% of the total

36 number of eggs laid in the 0-4-cm interval (Figure 3-10). There was not a significant difference between treatments (F= 0.05; df= 1,9; P= 0.8289), but a significant effect due to the depth was observed (F= 9.88; df= 1,18; P= 0.0056).

The treatment ∗depth interaction was not significant (F= 3.12; df= 1,18;

P= 0.0944). The mean number of eggs laid in 0-4-cm and the 4 to 24 cm depth intervals of the AT treatment were similar (Figure 3-11). More eggs were laid in the 0-4-cm interval than in the 4 to 24 cm depth intervals of the NT treatment (Figure 3-11).

Discussion

P. aequinoctialis females preferred to lay eggs in the MCT treatment more than in any other substrate. Beetles deposited more than 77% of their eggs in the MCT treatment in both choice arenas. When given the choice between artificial tunnels and no tunnels, females deposited a similar number of eggs in each treatment. In each choice arena, females deposited the highest number of eggs in the 0-4-cm sand interval for both treatments. However, the presence of tunnels largely influenced where most eggs were laid. For instance, in sand without tunnels most eggs were laid closer to the sand surface than sand with tunnels. When tunnels were present, eggs were laid in high numbers at the sand surface, but eggs were also consistently laid in the deeper depths. In the MCT treatment of the MCT versus NT arena, close to 40% of the eggs were laid in the sand intervals deeper than 12 cm. However, because the treatment ∗ depth interaction was not significant, it is difficult to determine how the two tunnel types influenced the distribution of eggs when compared to the NT treatment. In the AT versus NT arena, the treatment ∗ depth interaction was approaching significance and more eggs were being

37 laid deeper in the AT treatment than compared to the NT treatment. Therefore, further testing might have made this interaction significant.

Finally, because a larger number of eggs was laid in the MCT treatment than in the

AT treatment of the MCT versus AT arena, possibly the presence of stimuli associated with mole cricket-excavated tunnels appeared to influence beetle oviposition behavior. If only the presence of tunnels was important to the beetle for oviposition, then a similar number of eggs within each of these treatments would have been expected. In addition, because similar numbers of eggs were laid in the AT and NT treatments of the AT versus

NT arena, the presence of tunnels again did not appear to influence beetle oviposition. In this arena if the presence of a tunnel was important to females, a higher number of eggs laid in the AT treatment would have been expected. From these observations, the female beetles appeared to oviposit preferentially in Scapteriscus mole cricket tunnels than in artificially created tunnels or when no tunnels were present. Therefore, perhaps the females were detecting some cue within the mole cricket tunneled sand to direct oviposition. Lake (2000) found that females were not attracted to mole cricket eggs in a

Y-tube olfactometer so perhaps developing bioassays based on mole cricket cuticular hydrocarbons, anal gland secretions, or excretory products would be informative.

Table 3-1. Results of the contrasts procedure between depth intervals for both treatments. Depth intervals were designated as 1 = 0-4 cm, 2 = 4-8 cm, 3 = 8- 12 cm, 4 = 12-16 cm, 5 = 16-20 cm, and 6 = 20-24 cm. Contrast DF p- value 1 vs (2-6) 90 0.0301* 2 vs (3-6) 90 0.3341 3 vs (4-6) 90 0.9287 4 vs (5-6) 90 0.5449 5 vs 6 90 0.9914 *Significant at the α= 0.05 level

Figure 3-1. Plexiglas sandwich with black construction paper in place

Figure 3-2. No tunnel (NT) and Mole cricket tunnel (MCT) treatments with Plexiglas barrier in place

Figure 3-3. Placement of the artificial tunnel (AT) treatment in all oviposition arenas

Figure 3-4. General MCT versus AT arena before the beetle was released for oviposition

Figure 3-5. Oviposition arena and sand intervals within each treatment

200

180 MCT NT

160

140

120

100

80 Total no. of eggs

0 0-4 4-8 8-12 12-16 16-20 20-24 Depth (cm) Figure 3-6. Total number of eggs laid in each depth interval of the MCT and NT treatments

1.6 a MCT NT 1.4 ab 1.2 a

1 b b bb 0.8 b

0.6 transformed ls means estimate 0.4 c 0.2 c c c 0 0-4 4-8 8-12 12-16 16-20 20-24 Depth (cm)

Figure 3-7. Plot of the transformed least squares means estimates for each depth interval of the MCT and NT treatments. Different letters indicate significant differences between depth intervals of the same treatment at the α= 0.05 level

350

MCT AT 300

250

200

150 Total no. eggs Total

100

0 0-4 4-8 8-12 12-16 16-20 20-24 Depth (cm) Figure 3-8. Total number of eggs laid in each depth interval of the MCT and AT treatments

1.8 a MCT AT 1.6 ab 1.4 a bc 1.2 bc c 1 c

0.8

0.6 b bb ransformed ls means estimates t 0.4 bb

0.2

0 4-8 8-12 12-16 16-20 20-24 0 4?? Depth (cm)

Figure 3-9. Plot of the transformed least squares means estimates for each depth interval of the MCT and AT treatments. Different letters indicate significant differences between depth intervals of the same treatment at the α= 0.05 level

180

160 AT NT

140

120

100

60 Total no. eggs

0 0-4 4-8 8-12 12-16 16-20 20-24 Depth (cm) Figure 3-10. Total number of eggs laid in each depth interval of the AT and NT treatments

1.2

a AT NT 1 s a 0.8

0.6 a

0.4

transformed ls means estimate b 0.2

0 0-4 4-24 Depth (cm)

Figure 3-11. Plot of the transformed least squares means estimates for each depth interval of the AT and NT treatments. Different letters indicate significant differences between depth intervals of the same treatment at the α= 0.05 level

CHAPTER 4 INFLUENCE OF Scapteriscus abbreviatus EGG CLUTCH DEPTH ON THE LARVAL SURVIVAL OF Pheropsophus aequinoctialis

Introduction

The larvae of Pheropsophus aequinoctialis are specialized predators of

Scapteriscus mole cricket eggs in some parts of South America. Scapteriscus mole crickets deposit 25-60 eggs depending on female size and age in small, ovoid egg chambers. The egg chambers are located 9-30 cm underground depending on soil moisture and are closed off from their main tunnels (Forrest 1985). P. aequinoctialis first instars are very active and have longs legs enabling them to find the mole cricket egg chambers underground. Once the first instar penetrates an egg chamber it begins to feed and soon after will molt into a very different looking second instar. The second instar and all later instars have legs that are much shorter and a wider body when compared to the first instar. Hypermetamorphosis is the term used to describe the considerable changes that occur after first instars molt into the second instar of beetles with specialized predatory or parasitoid behavior. Finally, after all of the eggs are consumed, the larva has molted into the third and final instar and is confined to the egg chamber until the adult emerges and digs its way out.

In response to egg predation by P. aequinoctialis larvae, it is possible that

Scapteriscus mole crickets try to avoid egg predation by creating their egg chambers at depths which may hinder the ability of the first instars to find the eggs. It is also possible that P. aequinoctialis females try to increase larval survival by laying their eggs in mole

44 45 cricket tunnels, which have egg chambers located off from them. Based on the above assumptions, the objective of this experiment was to determine whether the depth of the mole cricket egg chamber influences the ability of P. aequinoctialis first instars to find mole cricket eggs.

Materials and Methods

To demonstrate whether a relationship exists between larval survival and mole cricket egg chamber depth, P. aequinoctialis first instars were released into sand-filled

PVC tubing containing S. abbreviatus egg chambers located at a range of depths. After a given period of time, the egg chambers were checked to determine whether beetle larvae located the egg chamber.

Scapteriscus abbreviatus Egg Chamber

PVC tubing, 5.08 cm (2 inch) in diameter and 9 cm long, was used as a mold to create the Scapteriscus abbreviatus egg chambers. The top of a 47-mm Millipore® plastic petri dish was placed at the bottom of the PVC tube. Four centimeters (81.03 cm3) of moist, autoclaved builder’s sand were placed into the PVC tubing and a 2 ∗ 3 cm oval depression was created in the middle of the sand using the end of a knife handle. A metal spatula was used to level the sides surrounding the depression. After 30 S. abbreviatus eggs were placed into the depression, the egg chamber was covered with 2 pieces of wooden Popsicle sticks, roughly 2.5-3 ∗ 1 cm, separated by a few millimeters

(Figure 4-1). One centimeter of sand was carefully placed on top of the sticks and leveled to complete the egg chamber (Figure 4-2). The egg chamber was removed by tapping the outside of the mold and by sliding the tubing off from the cylinder of sand.

Placing Egg Chambers over a Range of Depths

The previously created egg chambers were placed at the bottom of PVC tubing,

5.08 cm (2 inch) in diameter and of varying lengths, by sliding the tubes over the egg chamber molds. The egg chambers were secured in the bottom of the tube using duct tape. For each egg chamber depth, the PVC tubing was cut to a length at least 10 cm longer than the desired egg chamber depth. This space was needed for the egg chamber at the bottom, and so the larvae could not escape out of the top of the PVC tubing. For instance, when the egg chamber depth was located at 5 cm, the total length of the PVC tubing was 15 cm.

After the egg chamber was secured inside the tube, a string with 5-cm interval markings was lowered into the tube along the inner wall down to the surface of the egg chamber to measure the amount of sand to be added. Moist, autoclaved builder’s sand was placed carefully into the tube so the string remained at the inner wall of the tube.

After the desired depth of sand was added, the string was slowly pulled out and the sand was leveled off.

For each replication, 5 P. aequinoctialis first instars, no more then 7 days old, were placed onto the sand surface and the top of the tube was closed using black plastic, which was secured with a rubber band (Figure 4-3). Five larvae were released into the tubing to compensate for known mortality observed during laboratory rearing. However, only one larva will develop successfully due to larval fighting. PVC tubes were set up with egg chambers located at 5, 10, 15, 20, 25, and 30 cm deep. Six replications were completed for each egg chamber depth. Larval survival was determined by removing the duct tape and opening the egg chamber approximately 3 weeks after initially placing the larvae in the tube. A successful replication was determined by whether a beetle larva or pupa was

47 present in the chamber. An exact logistic regression analysis was performed on the proportion of successful replications for each egg chamber depth with SAS PROC

LOGISTIC (Derr, SAS Institute 2000), with depth as a continuous random variable.

Results

The percentage of successful replications for each chamber depth ranged from

33.3-100% (Figure 4-4). The lowest percentage of larval survival was when the egg chamber was located from 5 to 15 cm (Figure 4-4). From the exact logistic analysis, no detectable change in larval survival was apparent with the change in egg chamber depth

(P= 0.1235; Odds Ratio 95% C.I. [0.984,1.169]).

Discussion

The results of this experiment suggested that there was insufficient evidence to conclude that larval survival changed with the depth of the mole cricket egg chambers.

The percentage of surviving larvae tended to increase as the mole cricket egg chambers were located deeper, which might indicate that egg predation by P. aequinoctialis does not influence mole cricket oviposition depth. Furthermore, these results also might indicate that oviposition depth by P. aequinoctialis does not influence larval survival.

Instead, perhaps the location of oviposition relative to mole cricket tunnels may have a more important influence on larval survival.

The larval survival when the egg chambers were located from 5 to 15 cm was low and this may have been due to larvae drying out, escaping, or getting trapped in the apparatus. These seem to be the only plausible explanations because larvae could also find egg chambers at the lower depths, so it was not solely the experimental set up. If a relationship between egg chamber depth and larval survival exists, perhaps it could be demonstrated by completing more replications for each egg chamber depth. Completing

48 more replications may also decrease the likelihood that any particular egg chamber depth would have every larva developing successful, which was the case when the chamber was located 20 cm deep. Further investigation should consider completing more replications for each egg chamber depth and should place egg chambers at depths greater than 30 cm.

Figure 4-1. Egg chamber containing 30 S. abbreviatus eggs with Popsicle stick roof to hold the top sand layer

Figure 4-2. The completed egg chamber (left) and the PVC tubing, 5.08 cm in diameter, used to create the egg chambers (right)

Figure 4-3. Completed apparatus to test survival of P. aequinoctialis in a PVC tube 40 cm long containing an egg chamber located 30 cm deep

100

Percentage of successful replications 20

0 51015202530 Depth of egg chamber (cm) Figure 4-4. Percentage of successful replications when mole cricket egg chambers were located at depths ranging from 5 to 30 cm

CHAPTER 5 CONCLUSIONS AND FUTURE RESEARCH

The fecundity of laboratory-reared Pheropsophus aequinoctialis adults varied largely between individuals when fed the same diet over a 20-week period. Females began laying eggs on average a month after emergence. Two months after emergence females began laying fertile eggs and increased overall egg production. There was not a significant linear relationship between fecundity and the proportion of fertile eggs laid.

The explanation for the high level of variation in adult fecundity remains unknown from this study. Perhaps a relationship exists between body size and egg production that may better explain the differences in fecundity. In this study body size measurements such as elytron length and weight after emergence were not taken so this hypothesis could not be tested. In the regression analysis of fecundity and the proportion of fertile eggs, a linear relationship was approaching significance. A relationship must exist between fecundity and fertility because a female that is not fecund cannot be fertile.

Perhaps including more females into the analysis may have established a relationship, whether it was linear or not.

Oviposition behavior of P. aequinoctialis appeared to be directed towards the mole cricket tunnel treatment because significantly higher numbers of eggs were laid in this treatment in each arena that it was present. The presence of a tunnel did not appear to be an important stimulus for beetle oviposition because the number of eggs laid in the MCT treatment of the MCT versus AT arena was higher. Instead, the presence of a tunnel appeared to influence the distribution of the eggs throughout the arena. For instance,

51 52 females tended to lay a large number of eggs in the top intervals of all treatments, but when tunnels were present a considerable number of eggs was also laid in the deeper intervals. In the treatment without tunnels, females tended to lay a very small number of eggs deeper than 8 cm. Therefore, it appeared that the tunnels apparently were providing an area for females to move easily into the deeper intervals.

Rather than the presence of tunnels being important for oviposition, perhaps a cue associated with mole cricket-excavated tunnels is important for female beetle oviposition.

Lake (2000) attempted to determine whether adult females could detect mole cricket eggs and found that females placed in a Y-tube olfactometer containing mole cricket eggs were not attracted to the eggs. Detecting eggs that are in small, concealed chambers underground would be difficult for the beetles, so females probably would not have developed a searching behavior directed towards eggs. Instead, oviposition behavior may be directed towards compounds that occur in the tunnels such as mole cricket cuticular hydrocarbons, anal gland secretions, or excretory products. In the future, developing bioassays based on these compounds might then determine their influence on oviposition behavior.

Larval survival was not influenced by the change in mole cricket egg chamber depth. First instars are highly modified for host searching and the depth of the mole cricket chamber probably will not influence the ability of the larvae to find the eggs. The experimental set up did not exactly simulate a natural situation because egg chambers were located directly below the larvae, sand was packed uniformly, replications did not consider various soil types, and no barriers such as rocks were in the sand. However, the results did demonstrate that first instars have the ability to dig through many centimeters

53 of sand to find egg chambers. Completing more replications for each egg clutch depth may have provided the information necessary to determine whether egg clutch depth influences larval survival. In addition, completing replications at depths below 30 cm may have been able to show whether there is a depth at which the first instars cannot burrow down to. Another interesting experiment would be to determine whether a relationship exists between larval survival and the horizontal and vertical distance of female oviposition to the mole cricket egg clutch. This experiment might help to determine the influence female oviposition has on larval survival.

In conclusion, the observations from these experiments provided more of the information needed to understand the reproductive strategy of P. aequinoctialis.

Pheropsophus aequinoctialis females had the ability to lay large numbers of eggs, oviposition behavior was directed towards sand with mole cricket tunnels, and first instars could find egg chambers over the range of depths likely to contain mole cricket eggs clutches. These results could indicate that females lay a large number of eggs in or around mole cricket tunnels, which may increase the probability of their larvae finding mole cricket eggs. The evolution of host-directed searching behavior combined with a high reproductive potential could be an adaptation to increase the success of this species with a life cycle closely associated with another insect.

APPENDIX ADDITIONAL CHAPTER 2 FIGURES

120

100

60 No. Eggs No.

0 1 2 3 4 5 6 7 8 9 1011121314151617181920 W eek Figure A-1. Female 1, weekly total number of eggs laid after the start of oviposition

30 No. Eggs No.

0 1 2 3 4 5 6 7 8 9 1011121314151617181920 W eek Figure A-2. Female 2, weekly total number of eggs laid after the start of oviposition

54 55

120

100

60 No. Eggs

0 1 2 3 4 5 6 7 8 9 1011121314151617181920 W eek Figure A-3. Female 3, weekly total number of eggs laid after the start of oviposition

140

120

100

No. Eggs 60

0 1 2 3 4 5 6 7 8 9 1011121314151617181920 Week Figure A-4. Female 4, weekly total number of eggs laid after the start of oviposition

40 No. Eggs 30

0 1 2 3 4 5 6 7 8 9 1011121314151617181920 Week Figure A-5. Female 5, weekly total number of eggs laid after the start of oviposition

120

100

60 No. Eggs

0 1 2 3 4 5 6 7 8 9 1011121314151617181920 W eek Figure A-6. Female 6, weekly total number of eggs laid after the start of oviposition

No. Eggs 15

0 1234567891011121314151617181920 W eek Figure A-7. Female 7, weekly total number of eggs laid after the start of oviposition

30 No. Eggs

0 1 2 3 4 5 6 7 8 9 1011121314151617181920 Week Figure A-8. Female 8, weekly total number of eggs laid after the start of oviposition

160

140

120

100

80 No. Eggs 60

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 W eek Figure A-9. Female 9, weekly total number of eggs laid after the start of oviposition

120

100

60 No. Eggs

0 1234567891011121314151617181920 Week Figure A-10. Female 10, weekly total number of eggs laid after the start of oviposition

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BIOGRAPHICAL SKETCH

Aaron Scott Weed was born to Penelope B. Weed and Gary S. Weed in Waterville,

Maine. After birth he was raised in small town called Monmouth with his older sister

Melanie. While in Monmouth, Aaron attended Henry L. Cottrell Elementary, Monmouth

Middle school, and Monmouth Academy. At Monmouth Academy, Aaron concentrated on math and science courses (especially biology). While in high school, Aaron played 4 years of basketball, and 2 years of both baseball and soccer. He graduated third in his class of 44 and went on to Quinnipiac College to study biology. After one semester he transferred to the University of Maine, where he continued with biology as a major. It was not until his sophomore year of college that Aaron became extremely interested in entomology and botany. His first insect course, Introductory Entomology, introduced

Aaron to the world of insects. After this point, he began focusing his studies in this direction.

After collecting pitfall traps in the northern forests of Maine while working for the

State of Maine, he was exposed to the ground beetles, and took a fond liking to this group. Over the course of finishing his degree at UMaine, Aaron was also involved with

Potato IPM, pitfall and flight-intercept trap sorting, and identifying ground beetles to species level. For his undergraduate project, he identified all of the ground beetles caught in pitfall traps and prepared a formalized report on the diversity of ground beetles in an experimental forest exposed to different logging practices. At this point collecting, curating, and identifying insects really interested him. After wonderful advice given by

64 65 his undergraduate advisor, an aquatic entomologist, Aaron pursued graduate school to help broaden his knowledge and to help narrow his interests. However, with time his interests only grew larger and more diverse.