- Open Access
The effects of oviposition-site deprivation on Anopheles gambiae reproduction
© Dieter et al.; licensee BioMed Central Ltd. 2012
- Received: 30 July 2012
- Accepted: 23 September 2012
- Published: 16 October 2012
The African malaria mosquito, Anopheles gambiae, depends on availability of suitable surface water for oviposition. Short and long dry spells occur throughout the year in many parts of its range that limit its access to oviposition sites. Although not well understood, oviposition-site deprivation has been found to rapidly reduce egg batch size and hatch rate of several mosquito species. We conducted laboratory experiments to assess these effects of oviposition-site deprivation on An. gambiae and to evaluate the role of nutrition and sperm viability as mediators of these effects.
Anopheles gambiae adults (1–2 d old) from the G3 laboratory colony were assigned to the following treatment groups: oviposition-deprived (fed once and then deprived of oviposition site for 7 or 14 d), multiple-fed control (fed regularly once a week and allowed to lay eggs without delay), and age matched blood-deprived control (fed once, three days before water for oviposition was provided). Egg batch size and hatch rate were measured. In the second experiment two additional treatment groups were included: oviposition-deprived females that received either a second (supplemental) blood meal or virgin males (supplemental mating) 4 days prior to receiving water for oviposition.
An. gambiae was highly sensitive to oviposition-site deprivation. Egg batch size dropped sharply to 0–3.5 egg/female within 14 days, due to reduced oviposition rate rather than a reduced number of eggs/batch. Egg hatch rate also fell dramatically to 0-2% within 7 days. The frequency of brown eggs that fail to tan was elevated. A supplemental blood meal, but not ‘supplemental insemination,’ recovered the oviposition rate of females subjected to oviposition-site deprivation. Similarly, a supplemental blood meal, but not ‘supplemental insemination,’ partly recovered hatch rate, but this increase was marginally significant (P < 0.069).
Even a short dry spell resulting in oviposition-site deprivation for several days may result in a dramatic decline of An. gambiae populations via reduced fecundity and fertility. However, females taking supplemental blood meals regain at least some reproductive success. If mosquitoes subjected to oviposition-site deprivation increase the frequency of blood feeding, malaria transmission may even increase during a short dry spell. The relevance of oviposition-site deprivation as a cue to alter the physiology of An. gambiae during the long dry season is not evident from these results because no reduction in hatch rate was evident in wild M-form An. gambiae collected in the dry season in the Sahel by previous studies.
- Anopheles gambiae
- Dry season
- Hatch rate
- Vectorial capacity
Like most mosquitoes, Anopheles gambiae, the African malaria mosquito, depends on availability of suitable surface water for oviposition. However, during the dry season and dry spells during the wet season, larval sites may not be available for days, weeks, or even months in different environments[1–6]. Under such conditions, mosquitoes that develop eggs cannot lay them, a state hereafter referred to as “oviposition-site deprivation”, which differs from ‘egg retention’, a term applied also to an inseminated female which does not lay (some) developed eggs despite having access to a suitable oviposition site. The effect of oviposition-site deprivation on the physiology, behavior, and future life history of mosquitoes are scantly known, despite their possible ecological and public health implications. For example, the effects of oviposition-site deprivation on reproductive capacity, feeding rate, and longevity may correspondingly affect population size and disease transmission intensity during and following the period of deprivation. Moreover, not finding suitable larval sites may serve as a signal used by mosquitoes to switch from the typical reproductive state to reproductive depression in the dry season as previously described[8–14].
Only a few studies have evaluated the effects of oviposition-site deprivation on life-history traits. Reduced egg-batch size and hatch rate following oviposition-site deprivation was observed in Aedes aegypti, Ae. albopictus, Culex quinquefasciatus, An. maculatus, and An. pharoensis[7, 15–19]. In these studies, the duration of oviposition-site deprivation varied from a mere 2 days (An. pharoensis) to 10 weeks (Ae. albopictus and Cu. quinquefasciatus), however, statistical analysis was not always applied to the data. Both An. pharoensis and An. maculatus revealed increased sensitivity of anophelines to short-term oviposition-site deprivation (2–5 d) compared with culicine species (10–70 d), as measured by their egg batch size and hatch rate over shorter periods of deprivation.
Here we evaluate the effects of oviposition-site deprivation on the egg batch size and hatch rate of An. gambiae under laboratory conditions. We attempt to separate the effects of aging due to time and due to cumulative gonotrophic cycles from the oviposition-site deprivation itself and also evaluate the effect of sperm viability and the female nutritional state on the response to oviposition-site deprivation. Because this species experiences oviposition-site deprivation naturally, we hypothesized it would be less sensitive than other anopheline species previously examined. Our results suggest, however, that even short-term oviposition-site deprivation reduces egg batch size and hatch rate of An. gambiae and that nutrition is a key mediator of this effect.
On day seven, females to be assayed for oviposition (8–10 females/replicate cage of each treatment) were separated into individual 50 mL conical tubes lined with absorbent paper for egg laying and secured with netting on top to allow access to sugar pads. Males were also removed from all cages on day seven. Water for oviposition (10 mL of dechlorinated tap water) was added individually to the tubes four days after blood feeding (no oviposition-site deprivation), 11 days after blood feeding (short-term: 7-day oviposition-site deprivation), or 18 days after blood feeding (long term: 14-day oviposition-site deprivation). Control groups included females that were blood-fed regularly (once a week on days 4, 11, and 18) and those that were blood-fed only once (four days before water for oviposition was provided). Egg dishes were placed in the cages of the multiple-fed controls to allow the remaining females to lay eggs. Females that did not receive a blood meal on day 4 were considered blood-deprived (BD). These groups were fed once on day 11 or 18 and were provided with water for oviposition four days after feeding. Every treatment was replicated two or three times in each experiment (Figure1). A replicate is a set of all treatments in which females of a single cohort (same age) were housed in one cage/treatment during blood feeding. Oviposition assays were performed on 8–10 females per (replicate) cage in each time point (on day 8, 15, and 22, Figure1), thus providing a sample size of 16–30 per experiment. Hatch rate was measured for each egg batch that was laid.
Measures of egg batch size (EBS) and hatch rate were determined (see below). Females that died before their phenotype could be determined were excluded from analysis. The tubes were checked twice a day for three days; if eggs were laid, the female was killed and preserved and the appearance and number of eggs was recorded. Eggs that had sunk into the water were recorded separately and excluded from the count because they appeared abnormal and did not hatch in previous studies[20, 21]. Eggs were then placed in labeled petri dishes and submerged in water. Hatch rate was recorded by transferring newly-hatched larvae onto filter paper via a glass pipette and counting them. Egg dishes were checked twice a day for three days to complete the estimate of hatch rate. Females that did not lay eggs or died during the experiment were dissected to determine their insemination status. Non-inseminated females were excluded from the analysis. Mosquito body size was estimated using the measurement of wing length as previously described. Only females with at least one intact wing were measured.
The second experiment was carried out as stated above, with the addition of two treatment groups (each replicated twice). One treatment consisted of oviposition-deprived females that were provided with a second (supplemental) blood meal 4 days prior to receiving water for oviposition. The other treatment consisted of oviposition-deprived females, which received 100 4-day-old virgin males as supplemental mating. The males were kept in the cage for 3 days until the females were separated into oviposition tubes. A random collection of males from the same cohort and cages was placed with virgin females to assess their mating vigor. Virtually all virgin females were inseminated after 3 days (95%, n = 20). Individual females were separated and EBS and hatch rate were determined at the same time points as stated above.
This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal procedures were approved by the National Institutes of Health Animal Care and Use Committee (ACUC, Protocol ID: LMVR102).
Statistical analyses were performed using SAS 9.2 (SAS Institute, Cary, NC). The effects of the treatments on EBS and hatch rate were evaluated using mixed model ANOVA (implemented using PROC MIXED) because both fixed-effect variables (e.g., oviposition-deprived groups vs. non-deprived control) and random variables (e.g., experiment and replicate nested within experiment) were included. If significant differences were indicated between treatments overall, contrasts were used to specifically evaluate the effect of oviposition-site deprivation compared with a subset of controls. Thus, to assess the effect of 7-day oviposition-site deprivation on EBS, contrasts were made between that group and the non oviposition-deprived controls which were in their 1st and 2nd gonotrophic cycle (1 contrast), and between the oviposition-deprivation group and the age-matched blood-deprivation group which received their first and only blood meal 3 days prior to the water for oviposition (1 contrast). The third contrast compared the EBS of the 1st and 2nd gonotrophic cycle of the non oviposition-deprived controls. Because all females laid no (zero) eggs in some treatment/replicate combinations, resulting in non-estimable hatch rates, the variance component associated with ‘replicate’ could not be estimated in such cases. To determine differences in the percentage of females that laid eggs from different groups, log-linear categorical models were implemented using PROC CATMOD, which allows testing the same contrasts.
The effect of oviposition-site deprivation on the reproductive output of An. gambiae was determined by comparison of EBS and egg-hatching rate of females subjected to short (7 days) and long (14 days) oviposition-site deprivation with corresponding values of non-deprived females of the same gonotrophic cycle as well as those of the same (chronological) age but in the next gonotrophic cycle. Additionally, oviposition-deprived females were compared to females of the same chronological age and same gonotrophic cycle, which received their first blood meal 3 days before they were offered water for oviposition; the last group of females was deprived of a blood meal (blood deprivation). Unless otherwise stated, data obtained in the two experiments were analyzed together, treating experiments as random blocks (see Methods).
The effect of short-term (7 days) and long-term (14 days) oviposition-site deprivation on egg batch size (EBS) and hatch rate
(AIC) / (−2LL)a
OD-7 vs. Gon1 & Gon2
Gon1 & Gon2
OD-7 vs. BD-7
OD-14 vs. Gon1 & Gon3
Gon1 & Gon3
OD-14 vs. BD-14
OD-7 vs. OD-14
The proportion of oviposition-site deprived females that laid eggs (oviposition rate) was significantly reduced compared with non-deprived females, but the difference in mean EBS between the oviposition-site deprived and non-deprived females in those that laid eggs was minimal (Table1 and Additional file2: Figure S2). These results suggest that the overall effect of oviposition-site deprivation on EBS (including zero eggs) was mostly due to a lower oviposition rate rather than to a reduced number of eggs in those females that laid eggs. A high proportion of the oviposition-deprived females that did not lay eggs, especially those subjected to long oviposition deprivation, retained deformed brownish eggs that appeared to be undergoing resorption.
The roles of nutrition and sperm viability on egg batch size and hatch rate
The sensitivity of An. gambiae to short-term oviposition-site deprivation suggests that even short dry spells (measured in days) during the wet season can reduce reproductive success, especially because of lower fertility (hatch rate) augmented by lower fecundity (egg batch size) due to a lower rate of oviposition. Similar sensitivity was reported for other culicids including An. pharoensis and An. maculatus[7, 15–18]. High sensitivity to only a few days of oviposition-site deprivation is surprising for species that are often subjected to short (and long) dry spells under natural conditions, as is the case for An. gambiae. It is possible that a colony adapted to laboratory conditions has increased its sensitivity to oviposition-site deprivation because such deprivation is too rare in an insectary and it may also be related to other factors such as different blood source (chicken instead of human in our colony). However, the agreement across anopheline species (above), especially with the arid-adapted An. pharoensis, which was raised in the laboratory only three years prior to the study and was provided with human blood, suggests that the sensitivity to oviposition-site deprivation reflects the natural response of anophelines to such pressure.
However, oviposition-site deprivation was linked, as in most other studies listed above, with no (or limited) access to blood meals. The capacity to regain reproductive success by supplemental blood feeding suggests that, under natural conditions, mosquitoes may avoid the effects of oviposition-site deprivation by increasing their blood-feeding frequency. This hypothesis is consistent with the observations of gravid females that take additional blood meals[14, 23, 24]. Thus, malaria transmission may not decline (and might even increase) during a short dry spell. Additional studies measuring the blood-feeding rate of oviposition-site deprived An. gambiae females are required to clarify that hypothesis.
The importance of oviposition-site deprivation as a cue to alter the physiology of An. gambiae during the long dry season is not evident from these results. Similar to this study, Yaro et al. (2012) reported a reduced oviposition rate in field-collected (blood-fed, semi-gravid, or gravid) females that were provided with water for oviposition once they reached the gravid state (without experimental deprivation) in dry-season females as compared with their wet-season counterparts. Presumably these dry-season females blood fed while they were naturally deprived of oviposition sites. Unlike the present study, the egg batch size for females that laid eggs was also lower in the Sahel and a high frequency of brownish eggs was not observed. Although their study did not empirically measure hatch rate, the dramatic reduction in hatch rate was not observed during the dry season (Yaro, Alpha and Dao, Adama - unpublished data). These observations suggest that during the long dry season in the Sahel, altered female physiology modifies the effect of oviposition-site deprivation on their reproductive output. This hypothesis requires additional study, as well as the effects of oviposition site deprivation on survival and on the vectorial competence (susceptibility) with human pathogens, as was recently studied in Culex pipiens quinquefasciatus and West Nile Virus.
We thank Ms. Monica Artis for technical assistance during the laboratory work, Mr. Andre Laughinghouse and Mr. Kevin Lee for assisting with mosquito cultures, and Dr. Gwadz for facilitating the project. This study was supported by the Intramural Research Program of NIAID, NIH.
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