ABSTRACT Newly engorged larvae and nymphs of the lone star tick, Amblyomma anieri- canum (L.), were exposed to 9 treatments of pyriproxyfen in glass vials ...
Pyriproxyfen Effects on Newly Engorged Larvae and Nymphs of the Lone Star Tick (Acari: Ixodidae) W. A. DONAHUE, P. D. TEEL, O. F. STREY, AND R. W. MEOLA Department of Entomology, Texas A&M University, College Station, TX 77843-2475
J. Med. Entomol. 34(2): 206-211 (1997) ABSTRACT Newly engorged larvae and nymphs of the lone star tick, Amblyomma aniericanum (L.), were exposed to 9 treatments of pyriproxyfen in glass vials consisting of dosages of 4, 8, and 16 ju,g/cm2 for 7 d, 14 d, and continuous exposure periods at each concentration. Treatment of newly engorged larvae resulted in decreased molting, altered postmolt defecation, and nymphal survival with results being dose and exposure dependent. Molting inhibition ranged from 35.9 to 68.4%. Successfully molted nymphs were lethargic, exhibited altered defecation patterns, and were short-lived compared with untreated adults. By 65 d after treatment, cumulative inhibition of molting and hastened mortality of molted adults resulted in 82.6-100% control, depending on dosage and exposure. Treatment of newly engorged nymphs showed minimal to no effect on molting; however, adults were lethargic and displayed altered postmolt defecation patterns. Subsequent adult longevity was most dramatically affected with 87.9-100% control achieved by 82-84 d after treatment. Fecal patterns and survivorship were dose and exposure dependent. Estimates of subsequent feeding success of adults treated as engorged nymphs, show reduced capacities of attachment, engorgement and reproduction. KEY WORDS juvenile hormone analog, molting, mortality, Ixodidae, pyriproxyfen
DEVELOPMENT AND REPRODUCTION in hard ticks (Acari: Ixodidae) are believed to be regulated by juvenile hormones (Jaffe et al. 1986, Venkatesh et al. 1990, Sonenshine 1991). The model for endocrine and neuroendocrine systems in ticks is based on an understanding of these systems in insects. The lag of research progress in ticks, however, has left many elements and roles of these systems unclear (Binnington 1986, Diehl et al. 1986). For example, no juvenile hormone or juvenile hormonelike compounds have been identified conclusively in ticks (Sonenshine 1991), even though the observed effects of juvenoid compounds applied to ticks and the biochemical evidence to date support the presence of, and role for, similar substances in ticks. Even without conclusive evidence of the role of juvenile hormones, investigators have treated a variety of ixodid ticks including Boophilus decoloratus (Koch), B. microplus (Canestrini), Amblyomma hebraeum Koch, Hyalomma dromedarii Koch, and Rhipicephalus sanguineus Latreille, with juvenile hormone analogs, juvenile hormone mimics, and antijuvenile hormones. Among the effects observed were inhibition of oviposition, reduced oviposition, inhibition of egg hatch, delayed ecdysis, and hastened mortality occurring during ecdysis or postmolt (Soloman and Evans 1977, 1978; Hafez and Bassel 1980; Leahy and Booth 1980, Khalil et al. 1984; Connat 1988, Mansingh and Rawlins 1977). Responses were frequently found to be both dose dependent and temporally sensitive. Parameters for temporal sensitivity to juvenoid compounds are relatively undefined in ticks. Eval-
uation of insect growth regulator effects have been based primarily on continuous exposure of engorged males and females or nymphs to materials on substrates or by single topical or injection applications. Further, the persistence and fate of juvenoid compounds in ticks following single treatments or after removal from treated substrates is unknown. The lack of understanding of hormonal regulation in tick development and reproduction and of highly efficacious materials that would lend themselves to field application against ticks have largely slowed investigation into this potential area of tick control. Pyriproxyfen, 2-[l-methyl-2-(4-phenoxyphenoxy) ethoxy] pyridine, is a juvenile hormone analog that has desirable chemical and environmental properties (McLaughlin Gormley King Technical Bulletin 1993) which enhance its potential for tick control. Exposure of adults and eggs of the cat flea, Ctenocephalides felis (Bouch£), to pyriproxyfen will kill adults, prevent vitellogenesis or embryogenesis (Meola et al. 1993, Palma et al. 1993). The demonstration that pyriproxyfen, formulated as Nylar (McLaughlin Gormley King, Minneapolis, MN), can control larval fleas in pet loafing areas of lawns (Palma and Meola 1990) provides incentive for evaluation against ticks that share this environment. This report shows that pyriproxyfen residues in glass vials were effective in interrupting the molting and survival of the lone star tick, Amblyomma americanum (L).
0022-2585/97/0206-0211$02.00/0 © 1997 Entomological Society of America
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(continuously monitored by standard hygrothermograph), and a photoperiod of 14:10 (L:D) h. Technical pyriproxyfen was supplied by Mc- Three exposure periods were evaluated at each Laughlin Gormley King (Minneapolis, MN). Ace- treatment concentration. Exposure to pyriproxyfen tone used for dilutions was dried over anhydrous residues for 7 d, 14 d, and continuously was acsodium sulfate to remove any water from the ac- complished by transferring ticks to clean glass vials etone. The following 4 stock solutions were made with no pyriproxyfen residues at the prescribed exin volumetric flasks: (1) 93.28 mg (AI)/250 ml ac- posure time and returning them to the environetone, (2) 186.56 mg (AI)/250 ml acetone, (3) mental chamber. Fifty replicates of ticks were ex373.12 mg (AI)/250 ml acetone, and (4) acetone posed to each of the 9 (dosage-time) treatments only. Glass vials used for this bioassay were new and the continuous-exposure acetone controls. The 8-ml (2.0 dram) screw-top vials (17 by 60 mm) larvae were scored by vial (replicate) for the numwashed in soapy water, rinsed in distilled water, ber of molted and of live individuals. The vials and oven dried at >205°C for a minimum of 6 h. were also scored for total black and white postmolt The inside vial surface area was 22.64 cm2 and re- fecal spots deposited on the vial surface. quired 0.25 ml of acetone to coat the inside of the Nymphal Bioassay. Nymphs were placed onto vial completely. The 0.25 ml of stock solutions 1, chickens and allowed to feed naturally. Engorged 2, and 3 provided a final concentration of 4.0, 8.0, nymphs were collected, counted, and placed into and 16.0 fig of pyriproxyfen per square centimeter, plastic microcentrifuge tubes as previously derespectively. The 0.25 ml of solution was pipetted scribed for the larvae. Ten ticks per replicate were into a glass vial laid on its side. The vial was then transferred from the collection pan to microcenrolled to coat the inner surface evenly. Vials were trifuge tubes with a pair of soft teflon coated forrolled several times until the acetone was evapo- ceps. Forty replicates were transferred to glass virated and allowed to remain in a chemical hood als of each of the 9 (dosage-time) treatments and for 6 h to dry thoroughly. Acetone controls were acetone control. The nymphs were placed in the also treated in the same manner. All vials were pre- same environmental chamber as the larvae. Each pared on the same day for all treatments and the replicate was scored for the number of molted, treated and control vials were placed back in the number of live adults, and total postmolt black and original boxes and stored at — 15°C until needed. white fecal spots on the glass vial. Because of the high stability of pyriproxyfen over Excretory products of bloodmeal digestion and the range of temperatures in this study ( — 15- metabolism in ticks are heme-containing black de30°C), stability in organic solvents including ace- posits and guanine, a white crystalline material tone and low volatility (McLaughlin Gormley King (Balashov 1972). Alterations in the relative quanTechnical Bulletin 1993), the conditions used in tities of these products in tick feces may be indicthese studies were not anticipated to affect the fi- ative of digestive, metabolic, or excretory dysfuncnal concentration of active material. tion. Ticks used in these evaluations were from a colFour to 5 pairs of adult ticks ~A35 d after moltony of A. americanum originating from Brazos ing from treated nymphs were selected randomly County, Texas, and maintained at the Tick Re- from each treatment group and placed on a cow search Laboratory, Department of Entomology, in separate stockinette cells along with an addiTexas A&M University. Bioassays were performed tional cell containing 10 pairs of acetone control on engorged larvae and nymphs. Comparisons of adults. Ticks were monitored daily for attachment, means of selected variables were evaluated using feeding, mating, and fecundity success. the general linear model (GLM) and Tukey mean separation procedures (SAS Institute 1988) for Results and Discussion tests of statistical differences (a = 0.05) for both engorged larvae and engorged nymph bioassays. Larval Bioassay. The effects of pyriproxyfen exLarval Bioassay. Larval ticks were allowed to posure on newly engorged larval A. americanum feed on chickens. Engorged larvae that dropped are given in Table 1. Results show both a dosage within a 24-h period were collected and counted and exposure response as measured by the mean into plastic microcentrifuge tubes, 10 per replicate, number molted, percentage molted, postmolt fecal and capped. A hole was cut into the cap and cov- spotting, and postmolt nymphal survival on days ered with a fine mesh cloth. Ticks were transferred 29^30 and 65. from the collection pan to the tubes with a fine The mean percentage of engorged A. americanartists brush to avoid damage. Treated and control um larvae molting to nymphs in the acetone convials were removed from the freezer and allowed trol group demonstrated a high degree of success to warm to room temperature over several hours. and was significantly higher (F < 0.05) than any of Ten larvae were transferred from the centrifuge the pyriproxyfen treatments. Increasing dosage tube to each treated vial. Vials were covered with rates resulted in progressively lower percentages of a fine mesh cloth, secured with a screw cap man- nymphal molt. The increasing exposure times withufactured with a hole in the top, and placed in in each dosage level were not significantly different environmental chambers at 30°C, 70-80% RH (P > 0.05) from each other (Table 1), and means Materials and Methods
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Table 1. Effects of pyriproxyfen exposure (mean ± SEM) on 5 0 replicates of 10 newly engorged larval A. amer icanum per replicate Postmolt fecal spotting Trmt
% molted"
Total white
Total black
97.8 ± 0.008a
35.8
41.4
4 fig-1 d 4 fig-14 d 4 ju,g-Con
61.4 ± 0.022bc 56.6 ± 0.028bc 64.1 ± 0.026b
12.0 9.8 9.5
9.5 6.8 1.6
0.9 1.7 2.4 6.8
8 fig-7d 8 fig-U d 8 fig-Con
51.6 ± 0.023c 53.2 ± 0.026c 52.4 ± 0.025c
8.1 9.0 6.6
5.6 4.8 0.1
16 fig-7 d 16 fig-14 d 16 /j.g-Con
36.4 ± 0.024d 31.6 ± 0.025d 36.4 ± 0.026d
4.9 4.3 4.8
2.5 1.7 0.2
Control
Postmolt nymphal survival
W:B
29-30 d
ratio"
% alive
65 d
% alive"
% control"
0.027a
99.4
97.3 ± 0.012a
—
0.193ac 0.365ac 0.655b
66.1 65.4 81.6
16.9 ± 0.015c 8.1 ± 0.013d 0.6 ± 0.003b
82.6 ± 0.019b 91.7 ± 0.014ab 99.0 ± 0.009a
2.2 dt 0.331ad 3.4 it 0.359cdef 6.3 it 0.523b
53.1 43.2 80.9
10.5 ± O.Olld 4.9 ± 0.007bde 0b
89.2 ± 0.016ab 95.0 ± 0.012a 100a
2.4 jt 0.259ac 2.5 it 0.303ac 4.4 ii 0.404f
42.9 27.8 47.8
3.3 ± 0.006be 3.8 ± 0.006be Obe
96.6 ± 0.011a 96.0 ± 0.012a 100a
± d: i: d:
Trmt, treatments defined by concentration of active ingredient per square centimeter of inner vial surface with 3 exposure times: 7 d, 14 d, continuous (Con). W, white; B, black. Time interval is posttreatment of newly engorged larvae. Means within columns followed by the same letter are not statistically different (n = 50, P > 0.05); Tukey studentized honestly significant difference (HSD) range test (SAS Institute 1988). " Mean standard errors represent variation of treatment mean percentage in their decimal form.
at 8 fig/cm2 were not significantly different from exposure times of 7 and 14 d at 4 /xg/cm2. Postmolt nymphal survival was excellent for the acetone control group at both 29-30 d and 65 d posttreatment observations (Table 1). Survivorship in the control group at 65 d was significantly different (P < 0.05) from all treatment groups. In contrast, survivorship of nymphs from 29—30 d to 65 d declined substantially for all pyriproxyfen treatment groups. At 65 d, means within each dosage rate declined with increasing exposure, and these differences were greater at 4 and 8 /xg/cm2 than at 16 /xg/cm2. Statistical differences among treatment means for survival were supportive of the dosage and exposure effects of pyriproxyfen (Table 1). The inhibition of nymphal molting appears to be a key effect at these dosage rates and exposure times. The molting effects observed include incomplete apolysis or ecdysis, where the nymphal ticks remained entrapped within the larval exuviae. The old exoskeleton split transversely, but the emerging nymph could not disengage its appendages from the exuviae. These individuals were short-lived. Mortality, defined by no activity in response to repeated breath stimulation, and lethargy observed after the larval molt increased with dosage rate and length of exposure within the treated groups. Those individuals that successfully molted did not appear to be as active as those in the control group. Their behavior was lethargic and the nymphs congregated on the bottom of the vial as compared with the controls, where the nymphs were active and congregated at the top of the vial. The movement of the affected ticks was much slower and less precise than that of the control groups and in some cases the ticks were moribund, which was defined as incapable of locomotion but still capable of leg extension in response
to breath stimulus. Mortality increased for all the treated groups from molting through 65 d. Ticks in the control replicates had an average of 35.8 white fecal spots and 41.4 black fecal spots in the vials for a white:black ratio of 0.9 (Table 1). All the treated replicates had substantially fewer white and black fecal spots than the controls due in part to comparatively fewer survivors. The ratio of white to black fecal spots shows that black fecal spots progressively decreased with increasing dosage and exposure, and that these deviated from controls which have slightly higher numbers of black fecal spots relative to white. The means separation analysis showed that the ratio of white to black fecal spots in the control group was not significantly different (P > 0.05) from the lower exposure times: 7 and 14 d at 4 fig, 7 d at 8 fig, and 7 and 14 d at 16 /ug/cm2 (Table 1). The ratios of fecal spots observed at the continuous exposures at each dosage were significantly different from the control (P < 0.05). Among continuous exposure groups, the fecal ratios for 4 and 8 fig/cwr were not significantly different from each other (P > 0.05), and the fecal ratio at 16 fig/cm2 was not significantly different from the ratio at 8 fig/cm2, 14 d exposure (Table 1). Surviving nymphs from pyriproxyfen-treated cohorts appeared to desiccate. The appearance of these ticks suggested that there may have been a deficiency in water regulation or disruption of the ticks' ability to void guanine or other excretory products. This was most apparent in the difference between the fecal spotting by the ticks in the treated and control groups (Table 1). The behavioral change expressed as lethargy may preclude ticks from successfully questing, attaching to a host, and completing a blood meal under normal circumstances. The resulting mortality of a treated cohort of ticks would have a dramatic effect on that pop-
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A. Americanum
Table 2. Effects of pyriproxyfen (mean ± SEM) on 4 0 replicates of 10 newly engorged nymphal A. americanum per replicate Total no. ticks %
Postmolt adult survival
Postmolt fecal spotting " 49-51 d % alive
82-84 d
complete emergence
Total white
Total black
W:B •atio"
98.0 ± 0.006a
0
1.1 ± 0.037a
99.7
97.3 ± 0.007a
—
4 Mtf-14 d 4 /xg-Con
3 2 1
76.1 41.1 42.9 59.9
69.2
94.5:t 0.009a 95.2:t 0.008a 96.5:t 0.009a
87.9 ± 0.026a 95.8 ± 0.014bc 100c
4 0 1
0.051a 0.041a 0.054a 0.069a 0.074a 0.433b
11.3 ± 0.023b 4.0 ± 0.011c 0c
94.8:t 0.013a 94.8 :t 0.013a 96.2:t 0.011a
1.3: t 1.2 dt 1.5 :t 1.4 :t 1.2: t 3.6:t
98.4 93.4 95.1
8 M g-7 d 8 M g-14 d 8 /ng-Con
32.6 36.3 39.9 27.1 33.6 21.6
91.8 85.2 59.7
9.6 ± 0.021b 1.3 ± 0.006c 0c
89.6 ± 0.022bc 98.7 ± 0.011c 100c
16 /xg-7 d 16 /j.g-14 d 16 /xg-Con
97.3 =t 0.011a 93.8:t 0.012a 95.8:t 0.011a
0 0 0
27.5 24.8 8.9
1.6 d1 0.119a 1.5 :t 0.099a 9.5:t 1.265c
73.3 65.1 29.2
0.8 ± 0.004c 0.5 ± 0.003c 0c
99.2 ± 0.007c 99.5 ± 0.007c 100c
Trmt
Control 4 fig-! d
molted"
37.0 40.3 58.5 39.6 35.4 48.1
% alive"
% control"
Treatments defined by concentration of active ingredient per square centimeter of inner vial surface with 3 exposure times: 7 d, 14 d, continuous (Con). Trmt, treatment. Time interval is posttreatment of newly engorged nymphs. Means within columns followed by the same letter are not statistically different (n = 40, P > 0.05), Tukey studentized (HSD) range test (SAS Institute 1988). " Mean standard errors represent variation of treatment mean percentages in their decimal form.
ulation, especially with 3-host ticks. The combination of molting inhibition, changes in fecal elimination, and survivorship have not been demonstrated previously in a species of the Ixodoidea (Acari). Nymphal Bioassay. Effects of pyriproxyfen-exposure on newly engorged nymphal A. americanum are summarized in Table 2. Mean percentage of molt of nymphs to adults were consistently high across all treatments and were not significantly different (P > 0.05). There were 11 pyriproxyfen exposed ticks that were not able to emerge completely from their exuviae and died in this condition. These occurred only at the 2 lower dosages and were most pronounced at the shorter exposure periods (Table 2). Adults emerging from pyriproxyfen exposed nymphs exhibited a variety of characteristics that were not present among controls. These characteristics included lethargic locomotor activity, a gaunt physical appearance suggestive of dehydration, discolored cuticle, altered shapes in gut diverticula, and enlarged rectal sacs containing what appeared to be guanine. Although the percentage of molted ticks was not different among treatment and control groups, measurements of postmolt fecal spots showed that both guanine-containing white and heme-containing black spots of pyriproxyfen exposed ticks were substantially lower than values for untreated controls (Table 2). A dosage-exposure response was evident for these parameters among treated tick groups. Mean total white fecal spots tended to increase with increasing exposure time at each dosage level. Mean total black fecal spots tended to increase at 4 /xg/cm2 as exposure increased; however, at 8 /Ltg/cm2, black fecal spots at the continuous exposure were lower than at either of the shorter exposure times, and at 16 /u.g/cm2 the re-
sponse was a decline with each increase in exposure time. The lowest numbers of heme-containing fecal spots were counted for ticks with continuous exposure at the highest dosage. These relationships were reflected in comparisons of the ratio of white to black fecal spot means. Control and treatment mean fecal spot ratios were not significantly different from each other (P > 0.05), but were significantly different (P < 0.05) from those at 8 and 16 /Ltg/cm2 continuous exposure (Table 2), and the latter 2 treatments exhibited mean fecal spot ratios significantly different (P < 0.05) from each other. A dosage-exposure response was also exhibited in adult survival. Survival at 49—51 d showed that the most pronounced mortality occurred at 8 and 16 /ig/cm2 continuous exposures and that there were substantial declines in survival at all treatment levels >8 /ng/cm2 and 7 d exposure. Between 49-51 and 82-84 d after treatment, dramatic reductions in survival occurred across all treatment levels, whereas survival in the untreated control group was significantly higher (P < 0.05) (Table 2). Percentage of control of A. americanum with pyriproxyfen at 82-84 d postmolt was complete for continuous exposures at all dosage levels and exceeded 87% for the lowest dosage and shortest exposure period. Survival data were collected in this experiment under near optimal environmental conditions for this tick as suggested by the high percentage of survival in the untreated control group. Given the physical appearance and behavior of the treated ticks in this study relative to their fitness, it is doubtful that survival under field conditions would be equivalent. The fitness of treated adult ticks to attach, feed, and reproduce was examined by placing small numbers of randomly selected individuals surviving to 30 d postmolt in separate stockinette cells
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Table 3. Attachment, engorgement, and fecundity success of 30-d postmolt adult A. americanum L. placed in stockinette cells on a bovine after exposure of engorged nymphs to pyriproxyfen in glass vials
Trmt
No. pairs
Male
Control
Day 11 observations
Attachment at 72 h
Normal female CopuFemale engorgelation ment
Comment
1 M, 1 F dead
Fecundity % hatch egg Detach- No. ovi- No. masses ment position w/hatch Mean Range
10
8
15
Yes
Yes
9
13
12
89.2
1-95
4 /ng-7 d 4 Mg-14 d 4 /xg-Con
5 5 4
4 2
5 4 3
Yes Yes Yes
Yes Yes Yes
5 4 3
5 4 2
5 4 2
35.6 24.5 95.0
15-95 20-33 95-95
8 Mg-7 d 8 /xg-14 d 8 /ng-Con
5 5 5
5 0
4 5 4
Yes Yes Yes
Yes Yes
4° 4 0°
4 4
4 3
27.8 40.0
20-33 0-95
5 5 4 M, 5 F
3 3 0
3 3 2
Yes Yes No
Yes Yes No
3 0° 0
3
3
31.7
25-40
16 /Ltg-7 d 16 Mg-14 d 16 ^.g-Con
2 5
1 live male All dead
Treatments defined by concentration of active ingredient per square centimeter of inner vial surface with 3 exposure times: 7 d, 14 d, and continuous (Con). Trmt, treatment. All nonattached ticks were dead, except for 1 female still walking around inside the 4 figCon cell. Detachment of engorged females occurred 11-15 d postinfestation. " These cells contained partially fed females in the absence of live males 18 d after infestation.
by treatment on a bovine (Table 3). Proportionately fewer ticks that had received exposure to the higher dosages for longer periods were able to attach. Normal engorgement judged by coloration and rate of increase in size occurred at all treatment levels except that of the highest dosage and longest exposure period where ticks died in situ. Copulation was observed among ticks at each treatment level where males were able to attach and feed successfully. Most females that were able to complete engorgement and detach oviposited and nearly all egg masses produced larvae. Percentage of hatch was variable as indicated by the range of values across treatments (Table 3). With the exception of the 2 egg masses produced by ticks at 4 /Ltg/cm2, continuous exposure, the mean percentage of hatch for all treatments where larvae were produced was less than that measured for the control group. The percentage of hatch for the control group was lower than expected; 4 of the controls produced hatches of 95%, whereas the others were
