Acari: Ixodidae

Reduction in Egg Viahility Resulting from Infestations on Cattle of Hybridized Boophilus Ticks and B. microplus (Acari: Ixodidae) at Various Ratios RONALD B. DAVEY

AND LARRY

R. HILBURNl

Cattle Fever Tick Research Laboratory, USDA-ARS, Mission, Texas 78572

J. Med. Entomol.28(6), 763-769 (1991) The competitiveness of hybridized Boophilus males, which are 100% sterile, was compared to B. microplus (Canestrini). Hybrid larvae used in the study were the offspring derived by cross-mating B. annulatus (Say) males with-B. tnicroplus females. Cattle were infested with a total of 2,500 larvae at ratios of 5: 1 and 10: 1 (hybrid to pure strain). The reduction in egg hatch resulting from the hybrid males was 68 and 77.5% at the 5: 1 and 10: 1 ratios, respectively. Both treatment ratios produced egg sterility that were lower than expected, assuming purely random mating, suggesting that hybrid ticks were not as competitive as B. microplus males. At the 5: 1 treatment level, genotypic determinations based on isoenzyme analysis indicated that mate pairings involving hybrid males occurred 20-40% less frequently than expected, whereas matings involving pure-strain B. microplus males occurred twice as frequently as expected. At the 10: 1 treatment level, mate pairings involving hybrid males occurred 10-20% less frequently than expected, whereas pairings involving pure strain B. microplus occurred 2-4 times more frequently than expected. The results indicated that in a sterile hybrid male program, it would be necessary to increase the ratios of hybrids by '" 2-fold over the 5: 1 or 10: 1 ratios to achieve the 80-90% sterility expected, because of the decreased competitiveness of hybrid males.

ABSTRACT

KEY WORDS

Acari, competitiveness, Boophilus, sterile hybrid

value to the continued success of the Boophilus eradication program. Graham & Price (1966) reported that natural interbreeding may occur between populations of B. annulatus (Say) and B. microplus (Canestrini). Their report was the genesis for the idea that populations of these ticks might be controlled or eradicated if the hybrid offspring of these interbreedings were sterile or genetically altered in some way. However, it was not until 6 yr later that it was reported that hybrid males derived from cross-mating the two species were 99% sterile (Graham et al. 1972). Further studies determined that the sterility factor in hybrid males was caused by chromosomal incompatibility and that males remained sterile through three generations of backcrosses of hybrid females to pure-strain males (Newton et al. 1972; Thompson et al. 1981a,b). Once the sterility factor was established and the mechanism was described, it was critical to determine whether the sterilization process (hybridization) had any adverse effect on mating behavior or longevity of the sterile males (Knipling 1955). Investigations have indicated that hybrid m~les (type 2; B. annulatus male x B. microplus female) survive as long or longer and mate with as many or more females than pure-strain males (Davey et al. 1983). Davey (1986) reported that although type 1 hybrid males (B. microplus male x B. annulatus , Knipling-BushlandLivestockInsectsLaboratory,USDA-ARS, female) were not as competitive in mate seeking P.O. Box232, Kerrville,Texas78029. as either pure-strain male, type 2 males were equalIN THE EARLY1950s the potential of autocidal control technologies for management of pest populations came into prominence because of the phenomenal success achieved by using irradiated sterile screwworm flies, Cochliomyia hominivorax (Coquerel) (Bushland & Hopkins 1951, 1953; Baumhover et al. 1955; Lindquist 1955). Although autocidal control techniques have been successful with some pest species, they are impractical for controlling others. An a priori decision on the effectiveness of autocidal techniques against a given pest species is often difficult. However, the use of autocidal techniques should be given serious consideration where cultural, biological, mechanical, and other measures have minimal effect or where the use of pesticide is the primary means of reducing the population. The federally-funded eradication program that seeks to eliminate Boophilus spp. ticks from the United States is a prime example of a program that relies almost exclusively on the use of acaricides to eliminate the ticks. In addition, Boophilus ticks have few natural or biological enemies that reduce populations to any appreciable extent, and cultural and mechanical control measures, with the exception of livestock removal from infested premises to starve ticks, are largely nonexistent. Therefore, autocidal techniques, if effective, would be of great

PURCHASED BY THE U.

s.

DEPARTMENT

JOURNAL OF MEDICAL ENTOMOLOGY

764

ly competitive with either species. However, the effectiveness of the sterile male hybrid technology as a viable autocidal technique has been in some doubt more recently. It has been reported that assortative mating occurs between B. microplus and B. decoloratus (Koch) (Norval & Sutherst 1986). Furthermore, in another study it was reported that nonrandom mating occurs between B. microplus and type 2 hybrids (Hilburn et aI., 1991). These investigators reported that apparent nonrandom mating resulted from the hybrid males taking longer to reach mating maturity than B. microplus males, and real nonrandom mating occurred because hybrid males were not as competitive in establishing mating pairs as were the pure-strain males. These factors of assortative mating, nonrandom mating, and noncompetitiveness of hybrid males could have a highly adverse effect on the success of a sterile male program because the number of sterile hybrid ticks needed to overcome these adverse factors could be prohibitively high. The purpose of this study was to determine the degree of sterility that would be achieved by infesting cattle with various ratios (5: 1 and 10: 1) of hybrid to pure-strain ticks. Another objective was to determine the competitiveness of the hybrid ticks as compared to native ticks, if possible. The results of the study are essential to analyze the potential for success and the ratios required to eliminate native tick populations in a sterile male hybrid program. Materials

and Methods

Studies were conducted at the USDA-ARS, Cattle Fever Tick Research Laboratory, Mission, Tex. A total of 10 Hereford heifers weighing "='250 kg each were randomly assigned to four treatment groups consisting of two groups of three animals and two groups of two animals. The two groups consisting of two animals each were used as positive and negative controls. The positive control group, infested with only pure-strain B. microplus, provided an indication of the hatchability that would be expected from eggs laid by pure-strain female ticks in the absence of hybrids. The negative control was infested with only type 2 hybrid larvae. The type 2 hybrid larvae were the offspring that resulted from cross-mating B. annulatus males with B. microplus females. Thus, the negative control provided an indication of the hatchability of eggs laid by hybrid females, which have a reduced fertility when they are mated only to hybrid males that are totally sterile (Thompson et al. 1981a), in the absence of native fertile ticks. The remaining two treatment groups consisted of three animals each and were infested with either a 5: 1 or 10: 1 ratio of type 2 hybrids to pure-strain B. microplus larvae. Each calf within each treatment was individually stanchioned and held in a covered open-sided barn and infested with 500 larvae per day for 5 d

vol. 28, no. 6

consecutively (2,500 larvae total) of the appropriate type and ratio. Larvae used in the infestation were 14-21 d old at the time of infestation. Larvae of the different types were counted individually onto moist filter paper with a camel's-hair brush in the appropriate ratios, and papers with larvae were then placed on the midline of the back of each animal until larvae had successfully transferred to the host. When female ticks began to detach, all ticks from each calf within each treatment group were counted daily and their numbers were recorded. A random sample of ~50 female ticks per day for each host was held for oviposition purposes for nine consecutive days beginning on the first day of female detachment. Females were placed individually in coded 2-dram shell vials (17 by 60 mm) loosely fitted with cotton plugs and placed in an incubator at 25 ± 2°C, 92.5% RH, under a 12:12 (L:D) photoperiod. All females from each group were allowed to oviposit without disturbance for 8 d. Afterwards, the female was removed from the vial and placed in a 2-ml cryogenic vial, coded with the same information as the shell vial, and frozen in liquid nitrogen for later analysis. The eggs oviposited by each female were returned to the incubator. After 6 wk, when eggs had either fully hatched or it was evident no hatch would occur, the percentage hatch of each egg mass in each treatment was estimated by visual inspection to establish the overall egg hatchability in the four treatment groups. To estimate the overall ratio of hybrid to pure males on the host, irrespective of the males involved in mating, three random samples of 30-50 males were collected from each host in each treatment on the first, fifth, and last day when females were collected. The males from each host on each collection date were placed in 2-ml cryogenic vials, frozen in liquid nitrogen, and held for later analysis. The genotype of the ticks in the 5: 1 and 10: 1 ratio treatment groups was determined by two methods. The first method was used to determine the genotype of males that were mated to randomly sampled females collected during the nine consecutive days of female detachment and was made on the basis of the percentage hatchability of eggs of each individual female. If egg hatch from an individual egg mass was ~1%, then the male was considered hybrid (Thompson et al. 1981a), and if egg hatchability of the egg mass of a female was > 1%, the male involved was considered B. microplus. The second method for determining the genotype of ticks was electrophoretic analysis, which was conducted on each engorged female collected from each host on each day, and also on each randomly sampled male collected from each host on three separate days. Each tick was examined for enzyme mobility differences for glucose phosphate isomerase (PGI, E.G 5.3.1.9), and the anodally migrating form of aconitate hydratase (ACON-A, E.G

1991 DAVEY & HILBURN: REDUCED EGG HATCH CAUSEDBY HYBRID Boophilus TICKS 765

November Table 1.

Mean fetnale DUD'lbers,

eg8 hatchability.,

and

percentage reduction in egg viability resulting from infestattons of hybridized Boophilus licks and B. mlcroplus in various

ratios on cattle

n

FeHatio"

10:1 S:1 0:1 1:0

male ticks per host

% Egg hatch

766 720 710 712

20.8 28.S 92.3 0.02

Reduction of egg

G valuesb GH

Gp

GT

11 37*

212* 127*

223* 163*

hatch" 77.Sx 68.0y

a Means in this column followed by different letter are significantly different at the P < O.OS tested by a paired t test. b Test of daily samples within ratio classes. GH: test for homogeneity of samples; Gp: test of overall fit to defined ratio; GT: total. Values followed by * are significant at P < O.OS. C Hybrid (B. annulatus male x B. microplus female) to purestrain B. microplus ratio. Last two ratios indicate the positive (all B. microplus; 2,SOO total) and negative (all hybrid; 2,SOO total) controls, respectively.

4.2.1.3). The parental species B. microplus and B. annulatus have fixed allele differences at these loci (Sattler et al. 1986, Hilburn et al. 1989). Individual ticks were expressed of their blood meal and ground in ""0.2 ml of grinding buffer (0.01 M Tris'HCI, 0.001 M EDTA, pH 7.0) and the homogenate was absorbed into a wick cut from no. 1 filter paper. Proteins were separated by electrophoresing these homogenates on 12.5% starch gels prepared with N-(3-aminopropyl)-morpholine-citrate buffer (pH 7.0) (Clayton & Tretiak 1972) for 4 h at 35 mA and 4°C. The proteins were then visualized using histological staining techniques (Harris & Hopkinson 1976). Under these conditions, B. microplus has a single band for both of these enzymes, and hybrids have multiple bands. These differences made it easy to distinguish the pure-strain parentals from the hybrids. Data from each host within all four groups were pooled each day. The data on egg hatchability, mated males, mated females, and mating pairs observed for each of the nine days when females were collected and sampled were used as replicates within each treatment group. Daily hatch rates were subjected to the following modified Abbott's formula (Abbott 1925) to establish the percentage reduction of egg viability produced by the treatment ratios (5:1 and 10:1): REV

=

HPC - (HTG HPC

+

HNC)

x 100

where HPC is the percentage hatch in the positive control; HTG is the percentage hatch in the treatment group (5: 1 or 10: 1 ratio); HNC is the percentage hatch in the negative control; and REV is the percentage reduction in egg viability. These daily reductions in egg viability for each treatment group (5: 1 and 10: 1) were changed from percentages by arcsin transformation for analysis and subjected to a paired t test (Sokal & Rohlf 1969)

to determine if the reduction in hatch between the two treatments was significant. Data on egg hatchability from the two ratios (5 : 1 and 10: 1) were then subjected to a G test for goodne55-of-fit (Sokal & Rohlf 1969) to determine if the observed hatchability of eggs fit the expected hatchability. In addition, the data obtained for ratios of mated females, mated males, and types of mating pairs formed were also analyzed by a G test for goodness-of-fit (Sokal & Rohlf 1969), again using the nine collection days as replicates within each treatment group (5: 1 and 10: 1), to determine if the observed ratios fit the expected ratios for these factors. To determine the competitiveness of the sterile hybrid males as compared to native males, data were subjected to calculations described by Fried (1971) in which he reported a method of determining a point estimate of competitiveness for sterilized insects. The method is independent of the ratio of sterile to normal insects and can be calculated if the percentage egg hatch of the normal and sterile insects is known, as was the case in our study. Results The mean number of engorged females recovered from each animal showed little difference among treatments and controls, and ranged from 710 females per animal in the positive control to 766 females per animal in the 10: 1ratio treatment group (Table 1). These numbers indicated that the hybrid and B. microplus larvae were viable and survived to adulthood uniformly across the 10 animals tested. The mean hatchability of eggs produced by females ranged from 92.3% in the positive control to 0.02% in the negative control with the 5: 1 and 10: 1 treatment groups resulting in 28.5% and 20.8% egg hatch, respectively. Analysis of hatch for each of the 9 d (replicates) in the 5 : 1 treatment ratio gave significant G values (P < 0.05) for pooled (GP) and total (GT), indicating that something other than chance caused these nine samples to deviate from the 80% reduction in egg hatch that was expected. The daily egg hatch reduction ranged from 57.6 to 75.6% with an overall mean reduction in egg hatchability of 68%. Although the reduction was always less than the expected 80%, the magnitude of reduction, on a daily basis, was not uniform as evidenced by the significant G value for heterogeneity (GH) (P < 0.05). The 10: 1 treatment ratio produced a 77.5% reduction in egg hatchability, ranging from 69 to 83.9% and was significantly more effective (P < 0.05) in reducing egg hatch than the 5: 1 treatment (Table 1). Once again, significant G values for pooled (GP) and total (GT) showed that reduction in egg hatch was lower than the 90% reduction that was expected at a 10: 1 ratio. However, GH (heterogeneity) in this treatment was not significant,

Vol. 28, no. 6


766

Table 2. Expected "nd observed r"tios of Inale., feInale~, And mate pa_iring"_obtained hyhridi~cd Boophilu. and B. microplus at experimental hybrId to pure-stram rahoS of 5,1

evaluated Expected

Observed 5:1 Treatment

Mated females Mated males Random sampled Mated pairs"

males

males

10:1 10:1 10:1 100:10:10:1

Gp

GT

9 10

1S* 153*

22* 164*

23

178"

201"

24" 9

2 213"

26" 222"

42*

222*

264*

GH

group.

7.S:1 2.0:1 6.4:1 21:3:10:2

5:1 5:1 5:1 25:5:5:1

10: 1 Treatment Mated females Mated males Random sampled Mated pairs"

cattle with

G values"

Ratio· Factor

after inle.ting and 10,1

group.

8.7:1 3.1:1 11.0:1 82:9:26:4

a Hybrid (B. annulatus male x B. microplus female) to B. microplus. Test of daily samples within ratio classes. GH: test of homogeneity of samples; Gp: test of overall fit to the defined ratio; GT: total. G values followed by * are Significant at P < 0.05. C Ratios indicate the frequency of the genotypic combination of mating pairs (in order of appearance in the ratio) as follows: hybrid male to hybrid female; hybrid male to B. microplus female; B. microplus male to hybrid female; and B. microplus male to B. microplus female. b

reflecting the fact that the reduction in egg hatch over the 9 d did not differ in magnitude through time. Isoenzyme analysis conducted on mated females collected from the 5: 1 treatment group produced an overall ratio of 7.3: 1, ranging from 4.3 to 10:1 in favor of hybrid females over the 9 d sampling period (Table 2). Daily ratios favoring hybrid females were similar through time, as evidenced by the nonsignificant GR. However, significant GP and GT (P < 0.05) indicated that overall, the ratios deviated from the expected 5: 1 ratio. In the 5 :1 treatment, the overall genotypic ratio of males involved in matings (based on the egg hatchability of individual egg masses) was 2: 1 and ranged from 1.3 to 2.9:1 in favor of hybrid males over the 9 d. Ratios from each daily sample did not differ from each other (GH), but the significant GP and GT values (P < 0.05) resulted from the fact that the number of hybrid males did not reach the expected 5 : 1 ratio. In contrast to the 2 : 1 ratio of hybrid to B. microplus males involved in mating, isoenzyme analysis of nonmating males (random samples collected at three different times) produced an overall ratio of 6.4:1 hybrid to B. microplus males. Expected mating combinations and frequencies of each combination at a 5: 1 ratio of hybrid to pure-strain B. microplus are presented in Fig. lA, assuming that a 5 : 1 ratio for both sexes occurs and that mating is totally random. The nonsignificant GH for mate pairings showed that ratios of daily samples were consistent over time. However, the overall observed ratio of 21:3:10:2 differed significantly from the expected 25:5:5:1 ratio (P < 0.05), as evidenced by the GP and GT values (Table 2). Pairings involving hybrid males (first two numbers in the ratio) occurred 20-40% less frequently than expected, whereas pairings involving B. microplus males (last two numbers in the ratio) were twice as common as expected.

Isoenzyme analysis of mated females in the 10: 1 treatment group resulted in an overall ratio of hybrid to B. microplus that was 8.7: 1 and ranged from 2.6 to 18.3:1 over the 9 d when samples were taken (Table 2). Although the ratios on each day favored hybrid females, the magnitude of the ratios was not uniform, as evidenced by the significant GH (P < 0.05). The ratio began at about 10: 1 on the first day, rose to 18.3: 1 by day five, and then decreased to 2.6: 1 on day nine. Although some daily ratios were below the expected 10: 1 ratio, others were above the ratio, resulting in a significant GT (P < 0.05). The fact that some ratios were 10: 1 and some were < 10:1 led to a nonsignificant GP value. In the 10: 1 treatment group, the overall ratio of hybrid to B. microplus males involved in matings (determined by percentage egg hatch of individual females) was 3.1: 1 and ranged from 1.9 to 3.8:1 (Table 2). The nonsignificant GR reflected the fact that there was no difference among ratios over time. GP and GT values were both Significant (P < 0.05), showing that deviations from expectation always favored hybrid males, but they did not reach the expected 10: 1 ratio. By contrast, the ratio of randomly sampled males obtained at three separate times (nonmating) produced an overall ratio of 11 : 1 favoring hybrid males. The genotypic ratio of mate pairings and frequencies of each type for the 10: 1 treatment group are presented in Fig. lB. The observed ratio of mate pairings was 82:9:26:4 and differed significantly (P < 0.05) from the expected 100:10:10:1. Deviations from expectation resulted from the fact that mate pairings involving hybrid males were 10-20% less frequent than expected (first two numbers in the ratio) and mate pairings involving B. microplus males were 2.5-4 times more frequent than expected (last two numbers of the ratio). When data obtained in our study were subjected to calculations described by Fried (1971) to estab-

November

1991 DAVEY & HILBURN: REDUCED EGG HATCH CAUSEDBY HYBRID Boophilus TICKS 767

TREATMENT RATIO

(A)

(I-IYBRID~ NATIVE)

5: 1 EXPECTE MATING COMBINA ION AND FRE:QI E:NCY

I-IYBRID MALE X I HYBRID FEMALE COMBINATION FREQUENCY:

HYBRID MALE X NATIVE FEMALE

NATIVE MALE X HYBRID FEMALE

5

5

25

NATIVE MALE X NATIVE FEMALE

TREATMENT RATIO

(8)

(HYBRID: NATIVE)

10: 1 EXPECTE MATING COMBINA ION AND FREQl ENCY

HYBRID MALE X HYBRID FEMALE COMBINATION FREQUENCY:

HYBRID MALE X NATIVE FEMALE

100

10

NATIVE MALE: X HYBRID FEMALE

NATIVE MALE X NATIVE FEMALE

10

Fig. 1. Expected combinations of adult mate pairings and frequencies between hybridized Boophilus ticks and B. microplus infested on cattle at ratios of (A) 5:1 and (B) 10:1 (hybrid pure:strain).

lish the competitiveness of sterile males as compared to native males, we found that hybrid males had severely reduced competitiveness. At the 5: 1 and 10: 1 treatment levels, hybrid males were only 48 and 40% as competitive as native males, respectively. Discussion Results obtained in this study on egg hatchability in the two controls, 92.3% (positive) and 0.02% (negative), were consistent with the known hatching rates of pure-strain B. microplus and hybridto-hybrid matings, respectively (Thompson et al. 1981a,b; Davey et al. 1983, 1984; Davey & Cooksey 1989). The results obtained on reduction of egg hatchability at the 5: 1 and 10: 1 ratios suggested that characteristics associated with hybrid ticks caused reduction in egg hatch to be significantly

lower than the 80-90% that would be expected, assuming hybrids were equally competitive with B. microplus and random mating was occurring. At the 5: 1 and 10: 1 treatments, observed ratios of males sampled at three different times indicated that hybrid males were present in sufficient numbers, but their lack of competitiveness resulted in far fewer mate pairings involving hybrid males than would be expected if they were fully competitive with B. microplus males. Although the difference between expected and observed effectiveness of egg hatch reduction in both treatments may not appear extremely large ("'='12%), this difference may be much greater in terms of the reduced competitiveness of hybrid males (Fried 1971). Calculations indicated that hybrid males in this study were only 48 and 40% as competitive as native males at the 5: 1 and 10: 1 ratios, respectively. This suggests that it would be


768

necessary to release between 2 and 2.5 times as many hybrids as would be needed if hybrids were equally competitive to achieve the expected 80 or 90% reduction in egg hatch at the 5: 1 or 10: 1 ratio, respectively. Results obtained from isoenzyme analyses indicated that mate pairings involving hybrid males in both treatment groups occurred 10-40% less frequently than expected, despite the fact that ratios of non mating hybrid males on the host (random male samples) were nearly at expected levels. Conversely, mate pairings involving native males occurred 2-4 times more frequently than expected. It appeared that hybrid males simply did not engage in mating in proportion to their numbers. These results compare favorably with results of Hilburn et al. (1991), who reported that hybrid Boophilus males do not compete as well as B. microplus males for mates among either hybrid or B. microplus females. The eventual success of any autocidal program requires the ability to assess certain factors such as the competitiveness and longevity of sterile males (Knipling 1955). Until recently, reported research on competitiveness of hybrid Boophilus ticks indicated that hybrids were equally competitive with pure-strain Boophilus ticks (Davey 1986). In the absence of information to the contrary, predictive models (Osburn & Knipling 1982; Weidhaas et al. 1983) have operated on the assumption that hybrid males are equally competitive with native males. However, results of this study, as well as another investigation (Hilburn et al. 1991) call into question the assumption of equal competitiveness. Therefore, models and field tests should be reevaluated to reflect the reduced competitiveness of hybrid males. The lack of competitiveness of hybrid males indicated from this study does not necessarily preclude the eventual success of a sterile hybrid male technology. As long as the problem of reduced hybrid male competitiveness is known, it is not insurmountable. Solutions to the problem of noncompetitiveness in hybrids, such as increasing the number of hybrids released or selectively releasing hybrids into livestock gathering areas, may help offset the lack of competitiveness in hybrid males by increasing the number that reach hosts and subsequently compete with native males present on the host. Additional research is needed to establish the maximum capability for mass rearing and its cost effectiveness, to determine if it is feasible to rear and release hybrids in sufficient numbers at reasonable cost to reduce a native population of Boophilus ticks.

Acknowledgment The authors gratefully acknowledge the technical assistance of William Brozowski II, Claudio Castillo, Michael Moses, and Adolfo Pena without whose help this study would not have been possible.

Vol. 28, no. 6

Reference!> Cited Abbott, W. S. 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265-267. Baumhover, A. H., A. J. Graham, D. A. Hitter, D. E. Hopkins, W. D. New, F. H. Dudley & R. C. Bushland. 1955. Screw-worm control through release of sterilized flies. J. Econ. Entomo!. 48; 462-466. Bushland, R. C. & D. E. Hopkins. 1953. Experiments with screw-worm flies sterilized by x-rays. J. Econ. Entomo!. 44: 725-731. 1953. Sterilization of screw-worm flies with x-rays and gamma-rays. J. Econ. Entomo!. 46: 648-656. Clayton, J. W. & D. N. Tretiak. 1972. Amine-citrate buffers for pH control in starch gel electrophoresis. J. Fish. Res. Bd. Canada 29: 1169-1172. Davey, R. B. 1986. Mating competitiveness of hybrid Boophilus male ticks compared to pure-strain B. annulatus and B. microplus males (Acari; Ixodidae). J. Med. Entomo!. 23; 433-436. Davey, R. B. & L. M. Cooksey. 1989. Effects of prolonged exposure at low temperature on Boophilus microplus (Acari; Ixodidae). J. Med. Entomo!. 26; 407-410. Davey, R. B., R. L. Osburn & C. Castillo. 1983. Longevity and mating behavior in males and parthenogenesis in females in hybridized Boophilus ticks (Acari; Ixodidae). J. Med, Entomo!. 20; 614-617. Davey, R. B., R. L. Osburn & J. A. Miller. 1984. Ovipositional and morphological comparisons of Boophilus (Acari; Ixodidae) collected from different geographic areas. Ann. Entomo!. Soc. Am. 77: 1-5. Fried, M. 1971. Determination of sterile-insect competitiveness. J. Econ. Entomo!. 64: 869-872. Graham, O. H. & M. A. Price. 1966. Some morphological variations in Boophilus annulatus microplus (Acarina; Ixodidae) from northern Mexico. Ann. Entomo!. Soc. Am. 59: 450-452. Graham, O. H., M. A. Price & J. L. Trevino. 1972. Cross-mating experiments with Boophilus annulatus and B. microplus (Acarina: Ixodidae). J. Med. Entomo!. 9: 531-537. Harris, H. & D. A. Hopkins. 1976. Handbook of enzyme electrophoresis in human genetics. American Elsevier, New York. Hilburn, L. R., S. J. Gunn & R. B. Davey. 1989. The genetics of new world Boophilus microplus (Canestrini) and Boophilus annulatus (Say) in their possible contro!. Bull. Soc. Vector Eco!. 14: 222-231.

L. R., R. B. Davey, J. E. George & J. M. Pound. 1991. Non-random mating between Boophilus microplus and hybrids of B. microplus females and B. annulatus males and its possible effect on sterile male

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November

1991 DAVEY & HILBURN: REDUCED EGG HATCH CAUSED BY HYBRID Boophilus

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Received for publication 30 April 1991.

19 February 1991; accepted