Effect of the intensity of Hepatozoon gracilis parasitaemia on some biological aspects of Culex ( Culex ) pipiens ( Diptera : Culicidae )

The bean skinks were collected manually from Abu.Rawash, Giza, Egypt, separated according to size and investigated for H. gracilis infection. Intensity of parasitaemia was determined as slight (≤ 2% per 1000 counted erythrocytes), moderate (3-10% per 1000 counted erythrocytes) and high (11-20% per 1000 counted erythrocytes). Cx. (Cx.) pipiens L were reared under controlled laboratory conditions (20 + 1 °C, 60–70% RH and 10L: 14D photoperiod). Four groups of 3-4-day-old females, 50 individuals each, were starved 12 h prior to feeding. The first group was used to feed on parasite-free bean skink while the other groups were offered blood meals from slightly, moderately and highly infected lizards. The fully engorged females from each group were transferred separately to oviposition tubes and observed daily for longevity and reproductive capacity. Data were statistically analyzed by ANOVA using SPSS software, Ver., 15.0. The present study declared that the majority of naturally infected bean skinks were slightly infected. Generally the parasitic infections were costive to their host vector and this cost was manifested as a reduction in reproductive and transmission capacities. Preoviposition period was reduced by 15% and increased by 55% in case of mosquitoes fed on highly and moderately infected skinks, respectively, as compared to control. Fecundity was reduced by 48, 37 and 18% and fertility was reduced by 8, 3 and 6% in case of mosquitoes fed on highly, moderately, slightly infected skinks, respectively, as compared to control. No significant difference in the egg incubation period was observed between control and infected groups. Adult longevity was reduced by 40% in case of mosquitoes fed on highly infected skinks and increased approximately 2 folds in case of mosquitoes fed moderately and highly, infected skinks as compared to control. The present study suggested a decreased competence of Cx. (Cx.) pipiens in the hepatzoonosis disease circulation in nature when the infection intensity was high or moderate. On the other hand, the vector is a very strong candidate in the disease circulation in nature in case of slight intensity of infection.


INTRODUCTION
Apicomplexan parasites of the genus Hepatozoon have been described in many groups of terrestrial vertebrate and are especially prevalent in rodents and many families of carnivores (Smith, 1996).Numerous species of Hepatozoon have been reported to encompass a broad range of clinical disease in both wild and domestic animals (Rubini et al., 2006, Johnson et al., 2007, Breshears et al., 2009).The clinical syndrome resulted from Hepatozoon infection is known as hepatozoonosis.
Hepatozoonosis is an arthropod borne infection that affects numerous species Fatma H. Galal 58 including amphibians, reptiles, birds, and mammals (Smith, 1996).Hepatozoonosis syndrome varies clinically in different species; however, all share a common life cycle.This includes sexual development and sporogony within a hematophagous arthropod vector, ended by transmission to a vertebrate host and asexual development with merogony and gamontogony (Smith, 1996).Hepatozoonosis was diagnosed in many wild and domestic animals (Baneth et al., 2003, Potter et al., 2010).
Transmission of Hepatozoon spp. to vertebrate intermediate hosts usually occurs by ingestion of sporulated oocysts, which develop in the invertebrate definitive host.After confirmation of Ambylomma maculatum as the definitive host for Hepatozoon americanum (Mathew et al., 1998;Ewing et al., 2002) and determination of host preferences for immature A. maculatum (Barker et al., 2004), Ewing and Panciera (2003) suggested predation as a means of transmission of American canine hepatozoonosis.Forlano et al. (2005) demonstrated that A. ovale can be a vector of Hepatozoon spp. in Brazi.In addition, it was suggested that H. americanum, H. canis, as well as H. canis.likeorganisms were present and might cause disease in dogs in the southern U.S. (Johnson et al., 2008).However, infection via ingestion of cystozoites for H. americanum has only recently been documented supporting the concept of predation as a means of natural transmission (Johnson et al., 2008).
Because mosquitoes were considered as the most important medical insects, their biological aspects were investigated long time ago by several authors (Howlett, 1910, Galal, 1998, Dixit, 2001, Vinogradova, 2003).Culex pipiens was considered as cosmopolitan species, distributed all over the world all over the year.The efficiency of Cx. pipiens, as a transmitter of different human and animal pathogens, greatly depends upon their adaptability to harbor these pathogens without gaining detrimental effects.
The main objective of the present work was to evaluate the impact of intensity of Hepatozoon parasitemia on some biological aspects of the adult Cx. (Cx.) pipiens.

1-Field collection
The bean skink, Mabuya quinquetaeniata quinquetaeniata (Family: Scincidae), was collected manually from Abu.Rawash, Giza, Egypt.Collected lizards were separated according to size and then transferred as soon as possible to the laboratory and maintained at room temperature in small glass cages (35 x 35 x 35 cm) with sand beds and wire gauze tops.Water and small insects were supplied as food source for the lizards.The lizards were identified according to Mostafa (1997).

2-Parasite detection
To detect the presence of blood parasites, thin blood film from the peripheral blood of each lizard was taken by clipping off tail tips or leg according to the method described by Bashtar et al. (1984).The blood films were air dried, fixed in absolute methanol for 5 min and stained with 3% Giemsa's solution in phosphate buffer saline pH 7.3.The slides were washed in phosphate buffer saline pH 7.3 and examined under research light microscope (Olympus Optical Corp., Tokyo, Japan).Intensity of the parasitaemia was defined according to the actual number of erythrocytes examined (actually counted, not estimated from extrapolated partial counts or from the number of fields of view examined).Slight parasitaemia was defined as: ≥ 2% infected erythrocytes per 1000 counted erythrocytes, moderate parasitaemia was defined as: 3-10% infected erythrocytes per 1000 counted erythrocytes and high parasitaemia was defined as: 11-20% infected erythrocytes per 1000 counted erythrocytes.
Effect of the intensity of H. gracilis on some biological aspects of Culex (Culex) pipiens 59

3-Rearing technique of Cx. (Cx.) pipiens L
Cx. (Cx.) pipiens L was supplied from the Medical Entomologicaly Institute, Dokki, Giza, Egypt and colonized in the insectary of the Department of Entomology, Faculty of Science, Cairo University since July 2007 till now.Stock colony of the adult mosquitoes was maintained under laboratory conditions (20 + 1 °C, 60-70% RH and 10L: 14D photoperiod) for supplying adults of known ages.According to the method described by Adham et al. (2003), adult mosquitoes were reared in 35 x 35 x 35 cm cages.Each cage was provided with 10% sugar solution in a small Petri dish containing a piece of cotton soaked for feeding both newly emerged males and females.Sucrose solution was replaced daily to avoid fungal contamination.Blood meals were ensured by feeding upon domestic pigeon (Columba livia domestica).
Mosquitoes (3-4-days-old) were starved 12 h prior to the 1 st blood meal after adult female emergence.The gravid females were provided with plastic cups (7 cm diameter and 6 cm high) half filled with tap water for oviposition.Egg rafts were collected daily and transferred carefully to enamel pans (25 cm diameter and 9 cm depth) half filled with tap water and kept until the egg hatching.On hatching, 300 newly hatched larvae were transferred to a larger white enamel pan (40 cm diameter and 9 cm depth) containing about 500 ml tap water to avoid overcrowding.Larvae were fed daily on dry active yeast.Dead larvae, scum and exuviae were removed daily.Pupae were collected from breeding pans and transferred to the adult breeding cages.No attempts were made to control the light in the insectary room.However, during the working hours overhead fluorescent lamps were lighted.

4-The effect of the level of H. gracilis parasitaemia on some biological aspects of females Cx. (Cx.) pipiens L a-Reproductive capacity
Four groups of 3-4-day-old females, 50 individuals each, were starved 12 h prior to feeding.The first group was used to feed on parasite-free bean skink while the other groups were offered blood meals from slight, moderate and high infected lizards.The fully engorged females from each group were transferred separately to oviposition tubes (2 cm diameter and 5 cm depth) containing 1.5 ml of water and examined daily for oviposition.The preoviposition period, the number of eggs per female (fecudity), the incubation period of eggs and fertility (number of larvae hatched from each raft) were calculated.Each experiment was repeated four times under laboratory conditions during winter (20 + 1 °C, 60-70% RH and 10L: 14D photoperiod).

b-Adult longevity
Four groups of 3-4-day-old females, 50 individuals each, were used.Females were starved 12 h prior to feeding.The first group was allowed to feed on parasitefree bean skink while the other groups were offered blood meals from slightly, moderately and highly infected lizards.The fully engorged females from each group were transferred separately to vials and examined daily for survival.Dead females were removed and the number of survived females was recorded daily.The experiment was repeated four times under laboratory conditions (20 + 1 °C, 60-70% RH and 10L: 14D photoperiod).

5-Statistical analyses
Statistical analyses for biological data were carried out using SPSS computer software for Windows, Version 15.00 (Install Shield Corporation, Inc.).One way analysis of variance (ANOVA) and subsequent multiple comparison tests (LSD) were done to calculate the significance level between means.

Field studies
Out of 557 collected bean skink during 2009 from Abu.Rawash, Giza through 21 field trips, only 63 bean skinks (11%) were found infected with the blood parasite, H. gracilis.Out of 63 infected skinks, 18 were highly infected, 13 were moderately infected and 32 were tested as slightly infected skinks.The effect of the intensity of H. gracilis parasitaemia on some biological aspects of Cx. (Cx.) pipiens L

Reproductive capacity Preoviposition period
Table (1) shows that the preoviposition period was reduced by 15% and increased by 55% for mosquitoes fed on highly and moderately infected skinks, respectively, as compared to control.One way analysis of variance indicated over all significant difference between groups (df = 3, F = 45.03,P = 0.00).Multiple comparison tests revealed significant differences in the preoviposition period in case of mosquitoes fed on highly and moderately infected skinks as compared to control (P < 0.01).Meanwhile, no significant difference was observed in case of mosquitoes fed on slightly infected skinks as compared to control (P > 0.05).In addition, the difference in preoviposition period was significant between all infected groups as compared to each others (P < 0.01).

Fecundity (no of eggs/ female)
Table ( 1) showed that the number of eggs per female was reduced by 48, 37 and 18% in case of mosquitoes fed on highly, moderately and slightly infected skinks, respectively, as compared to control.One way ANOVA indicated over all significant difference between groups (df = 3, F = 5.39, P = 0.009).Multiple comparison tests revealed a significant difference in the fecundity of mosquitoes fed on highly, and moderately infected skinks as compared to control (P < 0.01).Meanwhile, no significant difference was observed in the fecundity of mosquitoes fed on slightly infected skinks as compared to control (P > 0.05).In addition, the fecundity of mosquitoes fed on highly and slightly infected skinks was significant (P < 0.05).

Incubation period
The data in Table (1) shows that the incubation period of eggs was not changed in case of mosquitoes fed on highly, moderately and slightly infected skinks, respectively, when compared to control.One way ANOVA indicated no significant difference between the three groups (df = 3, F = 0.00, P = 1).Multiple comparison tests revealed no significant differences in the incubation periods of eggs deposited by mosquitoes fed on highly, moderately, slightly, infected and control skinks (P < 0.05).

Fertility
The data in Table (1) shows that the fertility of mosquitoes fed on highly, moderately and slightly infected skinks was reduced by 8, 3 and 6%, respectively, as compared to control.One way ANOVA indicated an overall significant difference between all groups of mosquitoes (df = 3, F = 4.004, P = 0.015).Multiple comparison tests revealed significant reductions in the percentage of egg hatchability in case of mosquitoes fed on highly and slightly infected skinks as compared to control (P < 0.05).Meanwhile, no significant difference was observed in case of mosquitoes fed on moderately infected skinks as compared to control (P > 0.05).In addition, the difference in percentage of hatching of mosquito eggs fed on highly and moderately infected sinks groups was significant (P < 0.05).

Effect of the intensity of H. gracilis on some biological aspects of Culex (Culex) pipiens 61
Table 1: Effect of the intensity of H. gracilis parasitaemia on some biological aspects of Cx. (Cx.) pipiens.

Adult longevity
The data in Table (1) shows that the adult longevity was reduced by 40% in case of mosquitoes fed on highly infected skinks as compared to control.longevity was increased approximately 2 folds in case of mosquitoes fed on moderately and slightly infected skinks as compared to control.One way ANOVA indicated an over all significant difference between all groups of mosquitoes studied (df = 3, F = 34.79,P = 0.00).Multiple comparison tests revealed that longevity of adults fed on highly infected skinks was significantly reduced as compared to control mosquitoes (P < 0.05).Meanwhile, longevity was significantly increased in case of mosquitoes fed on lightly infected skinks as compared to control and mosquitoes fed on highly infected skinks (P > 0.05).No significant difference was observed between mosquitoes fed on slightly and moderately infected skinks (P < 0.05).

Percentage mortality
Fig.
(1) shows the daily mortality rate of female mosquitoes fed on slightly, moderately and highly infected skinks and control females.

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It was observed that the daily mortality rate increases gradually with the increase in the level of parasitaemia.The highest peak of mortality was observed by the 5 th (32%), 14 th (36.5%), 6 th (21.6%) and 8 th (69%) days post infection in mosquitoes fed on highly, moderately, slightly infected and control mosquitoes.Daily mortality curves of mosquitoes fed on highly and moderately infected skinks were reserved in higher rate than in mosquitoes fed on slightly and control skinks till the mortality of all females by the day 47 post feeding (Fig. 1).Mortality of mosquitoes fed on highly infected skinks started early by the 1 st day post feeding, reaching 40% by the 6 th day post feeding.The death rate fluctuates smoothly to 79% by the day 12 post feeding until all females diminished by the day 22 post feeding.Although the daily mortality of control females started by the 1 st day post feeding, a smooth curve was observed until all control females were diminished by the 47 th day post feeding.It was obvious that the death rate of mosquitoes fed on slightly infected skinks was more or less similar to that of the control ones (Fig. 1).

DISCUSSION
The present study suggested that the majority of naturally infected bean skinks were slightly infected.Little has been published on the infection rate of the lizard, Mabuya quinquetaeniata quinquetaeniata with the blood parasite H. gracilis.40 and 32% infection rates of the bean skinks with H. gracilis were previously recorded in Egypt (Bashtar et al., 1987 andIssa et al., 2000).This difference in the rate of infection may be attributed to the environmental conditions in the other localities (Issa et al., 2000, Nadler andMiller, 1984) or to the dissemination of the vector (Ball, 1967).The most probably controlling factor during this study was the abundance of the vector in the same vicinity of the host.
Generally parasitic infections are costive to their host vector and this cost is often manifested as a reduction in reproductive and transmission capacities.No significant differences were found in most parameters tested in case of mosquitoes fed on slightly infected skinks and control.Reproductive capacities of mosquitoes fed on highly and moderately infected skinks were significantly affected when compared to control.Significant reductions in fecundity, fertility and longevity were observed as compared to control.Combining these results with the results of Galal (1998), Adham et al. (2003) and Ebraheem et al. (2006 and2006), revealed that the decrease in fecundity was not caused by a reduction in blood meal size but mostly was a result of protozoon/ insect association.These results run parallel to the results presented by Hacker (1971), Hurd (1990) and Ferdig et al., (1993) who examined protozoon/ insect association in relation to fecundity for six strain of Aedes agypti infected with Plasomdium gallinaceum.The decrease in egg production might be attributed to the mean speed of oogenesis that was significantly retarded in infected mosquitoes (Carwardine and Hurd 1997).Also follicle resorption is one of the main factors contributing to malaria induced fecundity reduction in mosquitoes (Hopwood et al., 2001, Ferguson andRead 2002).However this relationship was contradicted by (Hammad, 1997 andCatarina et al., 2003) for Cx.(Cx.) pipiens and Cx.quinquefasciatus infected with Wuchereria bancrofti microfilaria as well as Ferguson et al. (2003) who reported that the mosquitoes fed on infected mice were more fecund.
No significant difference was found between the incubation periods of eggs of both control and infected females.Similar results were reported by Galal (1998)  Fertility was significantly reduced in case of mosquitoes fed on highly and slightly infected skinks as compared to control.Similar results were presented by Andreadis and Hall (1979) for Cx.salinarius infected with Amblyospora sp.Contradictory results were presented by Sweeney et al. (1989) who reported that there were no differences in fertility and fecundity of infected and control females.In another study, the authors didn't observe any effect of H. maturhensis on Cx. (Cx.) pipiens (Galal, 1998).
On comparing the longevity of control and infected groups we can conclude that the high infection greatly reduced longevity of mosquitoes (40%).However, moderate and slight infection resulted in 2 folds increase in adult longevity as compared to control.Inconsistent results were previously reported for An.albimanus infected with P. vivax (Chan et al., 1994) and for An.Gambiae infected with P. falciparum (Hogg and Hurd, 1997).
Consequently, mortality rates of mosquitoes fed on highly and moderately infected skinks were different as compared to slightly and control ones.These results are in agreement with those obtained by Klein et al. (1982) who stated that early increased mortality rate of the infected mosquitoes; An. dirus might be due to the rupture of the oocysts of P. gynomolyi.Findings confirming our results were reported for Glossina morsitans harboring Trypanosoma vivax, T. congolense and T. brucei by Moloo and Kutuza (1985), for Pseudolychia canariensis infected with Haemoproteus columbae by Rashdan (1991).The recorded increase in mortality is probably due to decreased efficiency in obtaining blood and increased feeding (Anderson et al., 2000).This relationship was disrupted by reports stating that mortality rate of infected mosquitoes are not differed from that of control ones (MacDonald, 1957).Furthermore, no significant difference in parasite burdens of dead mosquitoes was found.This finding could not detect a significant effect of infection on survivorship (Catarina et al., 2003).

CONCLUSION
Conclusively, this study described natural infection rate of the bean skink, the level of parasitaemia and its effect on some biological aspects of Cx. (Cx.) pipiens.The protozoan infection was, in general, costive to its mosquito vector.The reduced reproductive capacity, in addition to decreased adult longevity and increased mortality (in high and moderate infection) suggested decreased competence of the mosquito vector, Cx. (Cx.) pipiens, to share in the disease circulation in nature when the infection intensity was high or moderate.On the other hand, the vector is a very strong candidate to share in the disease circulation in nature in case of slight intensity of infection.This hypothesis could be strengthened by the previously obtained prepatent period (32 + 3days) after which the vector bite is infective as related to the vector longevity.

Fig. 1 .
Fig. 1.Effect of the intensity of H. gracilis parasitaemia on cumulative daily mortality rate of Cx. (Cx.) pipiens.