Journal of the American Mosquito Control Association, 22(4):609-614, 2006Copyright (C) 2006 by The American Mosquito Control Association, Inc.

FIELD AND LABORATORY COMPARISON OF HATCH RATES INAEDES ALBOPICTUS (SKUSE)

CHRISTOPHER J. VITEK AND TODD P. LIVDAHL

Clark University, Biology Department, 950 Main Street, Worcester, MA 01610

ABSTRACT. Laboratory experiments attempting to elicit a response based on a natural condition rely onthe assumption that the laboratory treatment accurately mimics field conditions. With Aedes albopictus(Skuse), laboratory experiments analyzing hatch rates assume that the laboratory stimuli resemble thosereceived by the eggs in field conditions. With the use of a colonized strain of Ae. albopictus, an analysis of thehatch rates comparing both field and laboratory settings was conducted. Additionally, hatch rates werecompared for mosquitoes exposed to regular, periodic hatch stimulation (as usually seen in laboratoryexperiments) and random hatch stimulation (as seen in the field). In both experiments, laboratory treatmentswere not found to differ significantly from the field treatments, indicating that experimental results achievedin the lab are relevant to field situations.

KEY WORDS Hatch stimuli, Aedes albopictus (Skuse), hatch rate

INTRODUCTION

Ecological experimentation in the field is moredesirable than attempting to replicate field con-ditions in a laboratory. It is difficult to control allnoncritical variables in the field, however, solaboratory experimentation becomes necessary.With laboratory experimentation, accuracy oflaboratory methods is essential for any well-designed experiment attempting to replicate fieldconditions. Without an accurate representation offield conditions, experimental results will alwaysbe subject to question. Ecological studies con-ducted in the lab should first verify that thetechniques and methods used accurately mimicfield conditions.Conducting hatch studies involving container-

breeding mosquitoes presents just such a dilemma.Even under ideal field conditions, many environ-mental variables cannot be controlled. Underlaboratory conditions, environmental factors canbe controlled to minimize their influence on thehatch rates of experimental egg batches. Themethod of hatching eggs in the laboratory maydiffer from the hatching stimulus that an egg willencounter in the field. Previous work has in-dicated that the hatch stimulus for container-breeding mosquitoes in the genera Aedes andOchIerotatus is a decline in the oxygen concen-tration after the eggs are submerged in a liquidmedium (Gjullin et al. 1941, Judson 1960,Barbosa and Peters 1969, Fallis and Snow1983). In the field, this oxygen concentrationdrop is caused by a bloom in the bacteriapopulation within a tree hole, brought on by aninflux of nutrients during a rainfall as nutrientsflow down the tree trunk into the tree hole(Walker and Merritt 1988, Walker et al. 1991). In

1present address: Florida Medical EntomologyLaboratory, 200 9th Street SE, Vero Beach, FL 32962.

the laboratory, the drop in oxygen concentrationcan be achieved by a variety of methods. Physicalmethods including artificially aerating the liquidmedium prior to egg immersion, chemical means,or biological means can all result in a drop in theoxygen concentration that can be detected by an

egg. These and other methods of hatch stimula-tion will result in different hatch rates underlaboratory conditions (Horsfall 1956, Novak andShroyer 1978, Livdahl et al. 1984). Hatching eggsusing the biological method of inducing a bacte-rial bloom may be the most similar to the hatchstimulus in the field. In the laboratory, bacteria inthe hatch medium have been shown to havea positive effect on egg hatch (Rozeboom 1934,Gillett et al. 1977). Presumably, this positiveeffect on hatch rate is the result of an oxygenconcentration drop in the medium, and the sameeffect occurs in the field. However, the responseto these stimuli in the laboratory may departfrom natural stimulus conditions.

There has been little field research conductedon hatching of Aedes albopictus (Skuse) eggs, nor

on the Aedes and Ochlerotatus spp. in general.One field study involved the inhibition ofhatching of Ochlerotatus triseriatus (Say) eggsby a high density of larvae (Livdahl and Edgerly1987), but for the most part field studies of hatchrates have been ignored. Additionally, there islittle research on the installment hatching of Ae.albopictus. Installment hatching, or delayed hatchof a fraction of eggs through successive stimuli,has been examined in a number of other tree holemosquitoes, notably Oc. triseriatus and Aedesaegypti (Linnaeus) (Gillett 1955). The majority ofthe hatching experiments involving Ae. albopictushave focused on the actual hatch stimulus(Matsuzawa and Kitahara 1966, Mogi 1976, Imai1993) and interactions with other species (Edgerlyet al. 1993).

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610 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 22, NO. 4

Our goal was to test laboratory hatchingmethods with the use of Ae. albopictus andcompare these methods to conditions an egg islikely to encounter in the field. By verifying thatthe hatch procedure under controlled laboratoryconditions is similar to field conditions, it ispossible to conduct further experiments in thelaboratory and hypothesize that results would besimilar to those in the field. Two aspects oflaboratory hatching were examined: the hatchmedium that is used in the laboratory, and thepattern of hatching stimulation the eggs receiveover time.The initial experiment compared the hatch

rates of eggs subjected to a variety of hatchmedia. The field hatch conditions consisted ofhatching eggs in both tree hole and tire dumpsettings. In the laboratory, the hatch mediaconsisted of a variety of nutrient broth concen-trations. The second experiment tested the in-fluence of 2 temporal patterns of hatch stimulus.In the laboratory, eggs are stimulated on

a regular, predictable basis over time, whereasin the field rainfall occurs in a much morerandom pattern. Eggs were subjected to stimulieither on a regular or random basis in thelaboratory to examine this effect. The actualprobability of stimulation was the same for boththe random and regular intervals between stim-ulation. The intervals of stimulation for therandom treatment were determined based onthe same frequency of stimulation as the regulartreatment. From these data, it is possible todetermine if laboratory methods of hatching eggsaccurately mimic field conditions. If it is de-termined that laboratory conditions can accu-rately reflect what is happening in the field, therelevance of laboratory conditions is establishedfor further experiments.

MATERIALS AND METHODS

For both of these experiments, a strain oflaboratory-reared Ae. albopictus was used. Thispopulation originated in the early 1990s fromlocations in North Carolina, and had been rearedfor many successive generations in the laboratory.Fertilized eggs from this population of Ae.albopictus were collected on wooden tomatostakes placed in the laboratory cages. Thesestakes were made from pine, and cut into 5-inch-long segments. Following oviposition, theywere cut into slivers, with similar numbers ofmosquito eggs on each sliver of wood creating anegg batch. Within 24 h of oviposition, these eggbatches were stored at a 16 h:8 h light:dark cycle,and kept damp in plastic vials until a sufficientnumber of eggs was obtained. For both experi-ments, the eggs were randomly divided into theexperimental groups, so the age of the eggs would

not affect the outcome (although age was in-cluded in the analysis).

Hatching medium." For the first experiment,egg batches were subjected to one of a variety ofnutrient broth concentrations, in an effort tomodify the strength of stimulus. Concentrationsincluded 1.0 g/liter; 0.5 g/liter; 0.25 g/liter, 0.1 g/liter, and 0.0 g/liter (distilled water). The nutrientbroth mixtures and distilled water were aeratedfor 30 min prior to submerging the eggs for 24 h.

Field stimuli were included for comparisons.Egg batches were placed in both tree hole and tirehabitats. Egg batches were contained within vialsthat had a fine plastic mesh on both ends of thevial, allowing liquid and nutrients to pass throughthe vial but not allowing the eggs or hatchedlarvae to escape. The tree hole vials were fixedhorizontally to the sides of tree holes in of 4beech trees immediately prior to a rainfall, justabove the waterline of the tree holes. The treeholes were selected based on the presence ofcontainer-breeding mosquitoes following previ-ous rainfalls. Four vials were each placed in 3different tree holes, and removed after 24 h. Asimilar procedure was used for the tire-dumpsetting, with 4 vials placed above the waterline in

of 2 discarded tires prior to a rainfall, andremoved 24 h later.

In all cases, hatched larvae were counted, andall unhatched eggs were dissected to determineviability, as indicated by the presence of a de-veloped larva. Once the total number of remain-ing viable eggs was determined, a hatch percent-age consisting of the number of hatched larvaedivided by the total number of viable eggs(hatched eggs plus remaining viable eggs) wasdetermined.Age of egg batches was suspected to influence

hatch rate. Because it was not possible to use eggsof uniform age throughout the experiment, agewas included as an independent variable. Treat-ment (hatch stimulation method) was randomizedacross the various ages. Within each egg batch,the age of the eggs was the same. Specific,planned comparisons were conducted with theuse of least-square means contrasts availablewithin JMP v. 4.0 software (SAS Institute, Inc.,SAS Campus Drive, Cary, NC).

Hatching pattern and frequency." The secondexperimental procedure was conducted by expos-ing eggs to sequential hatch stimuli, either atrandom intervals or at regular intervals. As withthe previous experiment, a hatch stimulus con-sisted of submerging the egg batch in an aeratednutrient broth medium consisting of 0.5 g/liter ofnutrient broth mixed with distilled water. Threedifferent frequencies for regular stimulation wereexamined: once every 3 days, once every 6 days,and once every 9 days. For the random hatchingintervals, this frequency (once every 3 days, once

every 6 days, and once every 9 days) was

DECEMBER 2006 FIELD AND LABORATORY COMPARISON 611

0.9

0.80.7

0.6

0.5

0.4=

0.3

0.2

0.1

0.0Dist.Water

b b b b a,b

0.1 0.25 0.5 1.0 tires treesg/liter g/liter g/liter g/liter

Stimulus type

Fig. 1. Mean hatch fractions for each of the hatchmedia (_+ SE). Each treatment was contrasted with allothers. Hatch fraction was calculated by dividing thetotal number of hatched eggs after the hatch stimulus bythe total number of viable eggs. Different letterscorrespond with significant differences in the meanhatch fractions (P < 0.05).

converted to a daily probability of being stimu-lated (0.33, 0.17, and 0.11, respectively). Thisprobability was then used to construct a dailyhatch schedule, using a draw of random numbersto determine days for stimuli on the randomstimulus groups. All experimental treatmentswere halted after 10 hatch stimuli. A medianhatch stimulus number was calculated for eachreplicate using a logistic regression. This medianis the stimulus number at which 50% of thedelayed-hatching eggs had hatched. A delayed-hatching egg is one that hatched after the 1ststimulus. Within each egg batch, cumulativehatch rates for each stimulus represented thedata points, with a hatch being a nominal value(either "y" or "n"). Because all eggs weresubjected to the initial stimulus at the same time,the logistic regression was applied only to eggsthat hatched after the first stimulus, counting thefirst stimulus as time 0, the second stimulus astime 1, etc. These batch medians constitutedobservations for comparisons of hatch delay forexperimental treatments.Pattern of frequency (random or regular

intervals) and the actual frequency of stimulationprovided nominal independent variables forcomparisons based on regression analysis. Aswith the previous experiment, the age of the eggbatches was included as a covariate, to attempt toreduce unexplained variation.

RESULTS

Hatching medium

Figure shows the average hatch fractions foreach of the treatments. Both hatching mediumand initial age of the eggs were significant factorsaffecting the hatch rate of the eggs, and there wasno evidence for an interaction between the 2

Table 1. Analysis of variance for the effects of initialage and hatching medium on the hatch rates of Aedesalbopictus eggs. The dependant variable of hatch rate

was calculated by dividing the total number of hatchedeggs by the total number of viable eggs for each

egg batch.

Source df SS F P

Age 2.50 53.91 < 0.0001Hatch medium (HM) 6 1.13 4.04 < 0.01Age HM 6 0.39 1.41 0.23Error 54 2.50

factors (Table 1). A detailed comparison of eachof the treatments with the use of a least-squaresmeans contrasts (JMP, v 4.0; SAS) indicates thatthere were differences in the hatch response to

some of the treatments (indicated on Fig. 1).Hatch rates in a broth medium were notsignificantly different at any concentrations (F

0.17, P 0.91). A regression analysis was alsorun on only the broth concentrations (0 g/literthrough g/liter) and was nonsignificant(F=1.97, P 0.17), confirming that there was

no difference in any of the broth treatments.

Stimulus pattern and frequencyThere was no significant difference in the hatch

rates on the first stimulus due to either frequencyor treatment (Fig. 2). The only factor that hada significant influence on the hatch rate duringthe first stimulus was the initial age of the eggs(Table 2). Age was also included in all subsequentanalyses for this reason.

After a critical stimulus number was calculatedfor each replicate, an analysis of covarianceindicated that there was no significant differencebetween the critical stimuli of the random or theregular treatments (Table 3). Stimulus frequencydid have a significant influence on the criticalstimulus. Egg age was also a significant factoraffecting the critical stimulus. There was no

Table 2. Analysis of variance for the first hatchfraction. The dependant variable hatch fraction wascalculated by dividing the number of hatching larvaeby the total number of viable eggs. The independentvariables are the stimulus pattern (subjecting the eggsto a hatch stimulus on a random or regular pattern),

stimulus frequency (how frequently the eggs aresubjected to a hatch stimulus), and the initial

age of the eggs.

Source df SS F P

Stimulus pattern 3.20 10 0.13 0.72Stimulus frequency 5.40 10 0.23 0.64Age of the eggs 2.11 10 8.78 < 0.01Error 34 8.16 10-

612 JOURNAL OF THE AMERICAN MOSQUITO CONTROL ASSOCIATION VOL. 22, NO. 4

0.45

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0.111 0.167 0.333

Frequency of Stimulation

O Regular

Fig. 2. Hatch fractions of the egg batches on thefirst hatch stimulus (_+ SE). Frequency of stimulationrepresents the probability of an egg batch beingstimulated each day, and the treatment (random orregular) represents whether the stimulation occurred on

a regular or random basis.

significant interaction between any of the vari-ables, and the interaction term was removed fromthe ANOVA analysis.

Figure 3 shows the critical stimulus numbergraphically. The extreme difference in the 0.33frequency means for random and regular treat-ments was due to median stimulus value of42.57 in the regular treatment. If this value wasremoved, the averages were much more similar(8.56 vs. 8.11).

DISCUSSION

Based on these results, it seems to be possibleto replicate field conditions in laboratory hatchrates accurately. Using the appropriate hatchmedium to replicate the desired setting is the firststep. The tree hole setting was not significantlydifferent from any of the laboratory media (anyof the broth concentrations or the distilled water).In addition, stimuli in tree holes seem to producea mean hatch rate intermediate between thedistilled water and the nutrient broth media.When attempting to replicate a tire stimulus, it

Table 3. Analysis of variance for the influences on themedian stimulus. The dependent variable medianstimulus was calculated with the use of a logistic

regression, and represents the predicted stimulus atwhich 50% of the delayed-hatching eggs would hatch.

The independent variables represent the stimuluspattern (stimulating the eggs on a regular or randombasis), stimulus frequency (the frequency at which theeggs are stimulated), the age of the eggs, and the firsthatch fraction (the hatch rate on the first stimulus).

Source df SS F p

Stimulus pattern 0.71 0.40 0.53Stimulus frequency 60.72 34.15 < 0.0001Age of the eggs 63.02 35.44 < 0.0001First hatch fraction 2.57 1.45 0.24Error 54 2.50

16

14

12

10

4

BRandom[

13 Regular

0.111 0.167 0.333

Frequency of Stimulation

Fig. 3. The median stimulus values for the differenttreatments. The bars represent the mean values for eachtreatment combination (+ SE). Random and regularrepresent the stimulus pattern, and stimulation frequen-cy is the probability of stimulation on a daily basis.

seems that using distilled water is the mostrealistic method tested. They both produceda similar mean hatch rate, and were notsignificantly different from one another. All ofthe nutrient broth concentrations had a highermean hatch rate than a tree hole setting (althoughnone of them were significantly different fromtree holes). Sex as a potential influence on hatchrates was not included in the analysis, but it maybe a relevant factor. Whereas males generallyhatch earlier and develop faster than females,there may be a sex specific response to thedifferent hatch treatments that warnts furtherstudy.The media tested are not the only possible

methods that can be used to hatch eggs. Othermethods described include using a chemical agent(usually ascorbic acid or bubbling nitrogen gas)to cause a reduction of oxygen concentration, orusing other biological means (such as a media ofcorn broth, rat chow, or mouse pellets) to cause

a bacterial bloom that will then lower the oxygenconcentration (Horsfall 1956, Mogi 1976, Novakand Shroyer 1978, Livdahl et al. 1984). Some ofthese methods, or a combination of the describedmethods, may provide a more accurate replica-tion of tree hole hatching. Treatment of eggsprior to submersion or stimulation can alsoinfluence the subsequent hatch rate (Horsfall1956). In our experiment, eggs were storeda minimum of 24 h following oviposition, andnot subjected to any wetting prior to actual hatchstimulation. Larvae presence may also alter hatchrates; some stimulatory effects have been found atlow densities, although as the density of larvaeincreases, egg hatch is inhibited (Livdahl andEdgerly 1987, Livdahl et al. 1984, Edgerly andMarvier 1992.The colony mosquitoes used in our experiment

had been reared under controlled conditions sincethe early 1990s. A colony strain of mosquitoes isexposed to environmental conditions that are

DECEMBER 2006 FIELD AND LABORATORY COMPARISON 613

vastly different from a wild population, includingbut not limited to fixed photoperiods, controlledhumidity and temperatures, and a readily avail-able food supply. Previous work has shown thatcolony strains rapidly adapt to laboratory condi-tions (Mogi 1976). In many cases, this is due toartificial selection that results from only subject-ing eggs from colony populations to hatchstimulus. Under these conditions, eggs that avoidhatching during the first stimulus are discarded,which results in a strong selection pressure,minimizing any hatch delay. In our colony, weroutinely subjected eggs to multiple hatch stimuli,in an attempt to avoid this specific selectionpressure. However, because of the length ofcolonization, it is conceivable that our colonyhas been subjected to other forms of artificialselection that may have altered their hatchresponse. As such, although our colony popula-tion responded similarly to both field andlaboratory hatch stimuli, it may be possible thatother colony strains or wild mosquitoes may notrespond in the same fashion.The regression analysis conducted on the

laboratory stimuli produced the same results asthe least squares means contrasts. Both indicatedthere were no significant differences between anyof the broth concentrations. Of the methodsexamined, .using a 0.5-g/liter nutrient brothmixture wag the closest to a tree hole setting,based on the average hatch fraction.The second aspect of replicating hatching in the

field is the pattern of stimulation. These dataindicate there is no difference between stimulatingthe eggs at regular or random intervals. In fieldconditions, stimulation via rainfall may occurrandomly, but in laboratory settings, regularstimulation is often more convenient. Interesting-ly, the stimulation frequency seems to have aneffect on the median number of stimuli requiredby delayed hatchers. This may indicate some sortof plasticity or ability of the mosquitoes torespond to environmental cues. It is known thatcontainer-breeding mosquitoes can detect andrespond to certain cues, including photoperiod,larval crowding, and a decrease in oxygen (pre-viously discussed as the actual hatch stimulus)(Shroyer and Craig 1983, Focks et al. 1994).However, laboratory populations that have beenreared for a high number of generations mayevolve different responses to hatching cues due toinadvertent selection pressures. Previous workwith Ae. albopictus has shown that a laboratorypopulation, initially collected from a forest set-ting and reared for an

extended length of time,developed an overall higher hatch rate (Mogi1976), and this could have resulted from theremoval of delayed-hatching eggs from thepopulation. For these and other reasons, recentlycaptured populations should be used to examineplasticity of egg responses to environmental cues.

The treatment of regular stimulation at a 0.33probability resulted in what appears to be a largedifference in the average median stimulus number(Fig. 3). However, this difference is primarily dueto replicate that had an estimated median valueof 42.57, approximately 4 times higher than anyof the median stimulus of any other treatment.This extreme value may have resulted froma failure to correctly identify nonviable eggs,which may have appeared as viable larvae ondissection. If this value is removed, the meanvalue is much more similar (8.56 for randomstimulation and 8.11 for regular stimulation).The influence of age on the hatch rates was not

surprising. The eggs of Ae. albopictus are resistantto but not immune to desiccation (Sota and Mogi1992a, 1992b). Increasing the time period be-tween oviposition and hatching increases theamount of time the egg is at risk of desiccation.As the time period between oviposition andhatching increases, the sensitivity of the egg to

a hatch stimulus may also increase to minimizethe egg's vulnerability to desiccation. Our resultsshow that increasing the time between ovipositionand the first hatch stimulus does result in a higherhatch rate. Desiccation resistance varies fromspecies to species, and different species mayrespond differently to increased aging of the eggsprior to hatching.

This information is vital for replicating fieldconditions in the laboratory. Given the inherentdifficulty of conducting field studies and thedifficulty of controlling all the variables in thefield, calibration of laboratory methods to obtainstimuli that are relevant to field conditions isessential. These results indicate it is possible toaccurately mimic field conditions in the labora-tory. In addition to the factors we studied, othervariables may influence the hatch rates of eggsand should be examined as well. Although itseems likely that other container-breeding specieswould respond similarly, wild populations of Ae.albopictus and other species may respond differ-ently than our colony strain of Ae. albopictus tothe hatch media and pattern of stimulation weexamined. A different treatment or combinationof treatments may be needed to mimic fieldhatching for other populations or species ofcontainer-breeding mosquitoes.

ACKNOWLEDGMENT

This research was supported by NIH grantsR15AI41191 and R15AI062712-01.

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