DOI: 10.5061/dryad.0zpc866w5
title: Quantifying species traits related to oviposition behavior and offspring survival in two important disease vectors

description: Animals with complex life cycles have traits related to oviposition and
 juvenile survival that can respond to environmental factors in similar or
 dissimilar ways. We examined the preference-performance hypothesis (PPH),
 which states that females lacking parental care select juvenile habitats
 that maximize fitness, for two ubiquitous mosquito species, Aedes
 albopictus and Culex quinquefasciatus. Specifically, we examined if
 environmental factors known to affect larval abundance patterns in the
 field played a role in the PPH for these species. We first identified
 important environmental factors from a field survey that predicted larvae
 across different spatial scales. We then performed two experiments, the
 first testing the independent responses of oviposition and larval survival
 to these environmental factors, followed by a combined experiment where
 initial oviposition decisions were allowed to affect larval life history
 measures. We used path analysis for this last experiment to determine
 important links among factors in explaining egg numbers, larval mass,
 development time, and survival. For separate trials, Aedes albopictus
 displayed congruence between oviposition and larval survival, however C.
 quinquefasciatus did not. For the combined experiment path analysis
 suggested neither species completely fit predictions of the PPH, with
 density dependent effects of initial egg number on juvenile performance in
 A. albopictus. For these species the consequences of female oviposition
 choices on larval performance do not appear to fit expectations of the
 PPH.  
description: <strong>Oviposition Experiments</strong>
 The oviposition responses to environmental factors for each
 species were conducted at the University of Southern Mississippi Science
 Park (31º 21’12” N, 89º 21’35” W). Experiments occurred outside in large
 wooden screen boxes (hereafter, arrays). Each array was 2.4 m long x 1.2 m
 high and 1.2 m wide, constructed with pressure treated lumber, and covered
 on the sides with no-see-um mesh (Phifer Inc., Tuscaloosa, AL); the top
 was covered with clear plastic sheeting, had a concrete bottom, with a
 door at one end. Each array had a small segment of PVC attached to one
 side affixed with a stocking net sleeve to introduce adult mosquitoes.
 Inside each array (n = 4) we placed 8 automobile tires (rim size 16”, mean
 size from [38]) vertically along the perimeter. Each tire was provisioned
 with one or more factors depending on the experiment and washed with a 10%
 bleach solution and rinsed before experiments. Each array received four
 sets of artificial potted plants placed in cups of sand between pairs of
 tires, to provide additional adult resting sites. Each array also held 20
 ml glass vials of 10% sucrose solution administered with a cotton wick as
 a food source for adult mosquitoes. Gravid females were introduced after
 all treatments, artificial plants, and sugar had been placed inside and
 the door sealed. <i>Pine detritus</i>.
 Senescent pine needles were collected from the Lake Thoreau Environmental
 Center, located 13 km from the USM campus (31°19’37.63” N, 89°17’25.22” W)
 and dried at 50 °C for ≥ 48 hrs. Dry needles were weighed and placed into
 tires containing 2000 ml of water and 200 ml of strained inoculum water
 (collected and homogenized from several undisturbed tires and strained to
 removed invertebrates), and allowed to exist outside for 3 d prior to the
 start of experiments. The range of pine detritus reflected values
 collected from natural tires [38]: 0, 14, 26, 38, 50, 62, 74, and 86 g.
 Tires were lined with brown paper as an oviposition substrate. We released
 20 gravid female <i>A. albopictus</i> into each array and
 after 48 hrs collected papers to count eggs. Four replicates were
 conducted, and detritus levels were haphazardly assigned to
 tires.            <i>Volume</i>. This
 factor was important for <i>A. albopictus</i> and <i>C.
 quinquefasciatus</i>, and thus the design was the same for both
 species. We used eight volumes that spanned the range found in [38]: 0.1,
 1.2, 2.4, 3.6, 4.8, 6.0, 7.2, and 8.4 L. The lowest volume corresponded to
 the lowest volume where larvae were collected [38]. Water added to tires
 consisted of a mixture of tap water and tire inoculum (prepared by taking
 a mixture of various detritus types and soaking in a 20 L bucket outside
 for several days). For each volume, we added 10 ml of inoculum for every
 100 ml of volume (e.g., 1.2 L would receive 120 ml tire inoculum).
 Because<i> Culex</i> lay eggs in rafts on the water surface,
 experiments involving that species did not include eggs papers. All other
 elements of design were the same as for pine detritus.
 <i>Canopy cover</i>. <i>Culex
 quinquefasciatus</i> trials involving variation in canopy cover used
 2000 ml of water with 200 ml inoculum in each tire. Variation in cover was
 achieved using segments of black no-see-um mesh placed on the array above
 each tire to decrease light penetration. We first measured light intensity
 next to two arrays at several times during each of a cloudless and
 overcast day using a hand-held light meter (Extech Light Meter Model
 401025, Nashua, NH). We used the mean of these two readings as the highest
 light level, then reduced the light by using more layers of black
 no-see-um mesh, with each layer reducing the level by ~50-60%. By using
 different layers of mesh, this allowed us to produce eight different
 levels of LUX (2400, 1200, 600, 300, 240, 180, 120, 60; higher values
 equate to more light). As above, all other elements of design were the
 same as for volume. <i>Statistical
 Analyses</i>. As oviposition responses to treatment levels were not
 independent (i.e., egg placement could affect the placement of other eggs
 in a tire) we used a mixed model randomized block design for each species
 and treatment, separately using PROC MIXED in SAS. For the egg oviposition
 experiments we included array as a random factor in the analysis. To meet
 assumptions of normality and homoscedasticity, the number of eggs of
 <i>A. albopictus</i> were log<sub>(x+1)
 </sub>transformed for volume. For <i>C.
 quinquefasciatus</i>, we used a 1/(x+1) transformation of egg rafts
 for volume, and log<sub>(x+1)</sub> for canopy cover.
 Differences in egg or raft numbers among treatment levels were identified
 using a Tukey-Kramer HSD post-hoc test for multiple comparisons. All
 analyses were performed using SAS. <b>Larval
 Survival Experiments</b> To assess the effects of
 our field parameters on larval survival, we also conducted laboratory
 experiments for both species using the same factors as those for
 oviposition. In all cases, we used black rubber bowls made of recycled
 tires (Miller Manufacturing, Glencoe, MN) to rear larvae to mimic the
 environment of tires. Bowls were kept in environmental chambers set at 27°
 C on a 14:10 light/dark schedule. For each replicate within a trial 90
 1<sup>st</sup> instar <i>Aedes albopictus</i> were
 used whereas replicates for <i>C. quinquefasciatus</i> used 55
 1<sup>st</sup> instars; these numbers reflect the mean number
 of larvae per tires from field observations [38]. In all cases, we used
 pupation as a surrogate for survival. The experiment lasted until all
 larvae had pupated or died. <i>Volume</i>.
 The treatments consisted of rubber bowls filled with different volumes of
 water as for oviposition experiments (0.1, 1.2, 2.4, 3.6, 4.8, 6.0, 7.2,
 and 8.4 L). Small bowls (2.5 L) were used for the two lowest volumes,
 whereas larger bowls (8.5 L) were used for the remaining volumes. Each
 bowl was filled with 95% by volume RO water and 5% by volume inoculum.
 After the water was placed in the bowls, 0.25g of freeze-dried animal
 (crickets, <i>Gryllodes sigillatus</i>, Fluker Farms, Port
 Allen, LA) detritus was placed in each bowl to provide food for developing
 larvae. Bowls were left in incubators for three days to encourage the
 growth of the microbial community before the addition of larvae.
 <i>Pine detritus</i>. Senescent pine needles were
 obtained from Lake Thoreau and dried at 50 °C for 48 hrs before
 establishing treatment levels identical to those used in the oviposition
 trials (0, 14, 26, 38, 50, 62, 74, and 86 g). Detritus was placed in
 individual 2.5 L bowls with 2.0 L RO water and 0.2 L tire inoculum
 strained with a 150-micron sieve. Detritus sat for 48 hrs before larvae
 were added. Larvae <i>A. albopictus</i> densities were
 identical as in the volume levels. <i>Canopy
 Cover</i>. Small bowls were filled with 2.0 L of water (1.95 L RO
 water, 0.05 L inoculate water). The highest light level within incubators
 registered at 1500 LUX and each subsequent layer of no-see-um mesh saw a
 decrease in light by ~50% (1500, 750, 375,180, 90, 45, 20, 10 LUX). Thus,
 this gradient of light levels was proportionally similar to but lower than
 those used in the oviposition trials. Detritus, soaking duration, larval
 <i>C. quinquefasciatus</i>, and other factors were identical
 as in the volume levels. <i>Statistical
 Analyses</i>. We assessed differences among treatment levels for
 mosquito survival using 1-way ANOVA. To meet assumptions of normality and
 homoscedasticity, survival of <i>A. albopictus</i> was square
 root +1 transformed for volume. For <i>C.
 quinquefasciatus</i>, we used a log<sub>(x+1)</sub>
 transformation for survival for volume and canopy cover. Differences in
 mosquito survival and oviposition among treatment levels were identified
 using the Tukey-Kramer HSD post-hoc test for multiple comparisons where
 applicable. <b>Linking oviposition to life
 history and survival</b> We performed a combined
 experiment to test the hypothesis that oviposition responses by females
 have consequences for life history and survival of progeny. Specifically,
 we repeated the oviposition experiment for each species but crossed each
 environmental factor within each array. For <i>A.
 albopictus</i>, we used three volumes (2.4, 4.8, 8.4 L) crossed with
 three levels of pine detritus (0, 14, 62 g). We excluded the 0 g pine
 detritus by 2.4 L volume, leaving us eight total combinations. For
 <i>C. quinquefasciatus</i>, we used three volumes (0.1, 2.4,
 6.0 L) and canopy cover (10, 750, 1550 LUX). Here, we excluded the 0.1 L
 by 10 LUX combination. Values chosen for both species generally elicited
 the highest oviposition responses of females during the separate
 oviposition trials (see Results). Because we planned to quantify different
 life history traits, we used 40 females during these trials to generate as
 many eggs as possible. We used the same general methods as in the separate
 oviposition trials (e.g., artificial plants, oviposition water, sugar).
 After 48 hrs, we counted eggs and rafts as before, but unlike the
 oviposition trials, here we returned larvae to the arrays and followed
 their development. Specifically, we hatched eggs of both species and
 placed offspring in 8.5 L rubber bowls within each upright tire. This was
 done to facilitate the identification and removal of pupae but to maintain
 the same microenvironment as the tire. <i>Aedes</i> egg papers
 were kept out of water for one week to allow for embryonation, whereas
 <i>C. quinquefasciatus</i> eggs, which hatch within 24 hrs,
 were immediately placed into bowls after counting emerging larvae. In all
 cases, we used the number of hatched larvae added to bowls to reflect
 female reproductive output. For treatment combinations that lacked food
 for developing larvae (e.g., all <i>C. quinquefasciatus</i>
 treatments, no pine treatment levels for <i>A.
 albopictus</i>), we added 0.25 g of crickets to each bowl. We ran
 four replicates of each species with two replicates run for each species
 during each trial (two trials were run to achieve our four replicates per
 species). All bowls were monitored every other day for the presence of
 pupae, which when present were removed and isolated in 0.25-dram shell
 vials until they eclosed. Adults were then sexed, identified to species,
 and then dried at 50º C for &gt; 48 hrs and weighed to the nearest
 0.0001 mg using an XP2U ultra-microbalance (Mettler Toledo Inc., Columbus,
 Ohio). For each bowl, we determined the mean female weight (mg), female
 development time (from egg to adult), and percent survival (males were
 excluded from weight and development times given that they are not the
 main driver of population sizes in mosquitoes as they don’t lay
 eggs). Oviposition patterns were linked to mosquito
 life history (female mass, development time) and survival using path
 analysis, which can test for the effects of multiple independent variables
 on multiple dependent variables. In path analysis, unlike multiple
 regression, a variable may be both dependent (i.e., affected by other
 variables) and independent (i.e., affecting other variables). Path
 coefficients, like standardized regression coefficients, quantify direct
 effects on a dependent variable caused by variation in an independent
 variable, independently of direct effects of other independent variables.
 The importance of a path was evaluated by testing the fit of reduced
 models in which paths are removed, relative to a full model. Reduced
 models were compared to the full model using goodness-of-fit
 χ<sup>2</sup> tests (PROC CALIS). We hypothesized three sets
 of paths based on the relationship between oviposition and larval survival
 in explaining life history parameters and survival:  a Combined Model
 (Fig. 3A), a Larval Model (Fig. 3B), and an Oviposition Model (Fig. 3C).
 The Combined model served as the full model, in which all variables were
 connected to all other variables. Moreover, if this model was supported in
 any way, it would suggest that both oviposition (i.e., number of hatched
 larvae) and larval survival and life history traits (female mass and
 development time) were both affected by habitat factors (i.e., ecological
 filters are consistent and important for both life stages). The Larval
 model contained direct effects of environmental factors on survival,
 female mass, and female development time. If this model was supported then
 it would suggest that habitat parameters largely worked to affect larval
 traits, but that egg laying was less important. The Oviposition model
 contained only the direct paths between environmental factors and the
 number of hatched larvae produced, and if supported, would suggest that
 filters only acted on females for each species. After analyzing the
 Combined model, paths were then removed in a systematic way, and if
 removing a link did not decrease the fit of the model, then it was assumed
 that the link was unimportant in explaining variation in the dependent
 variable. If this occurred, the path was eliminated and the next path was
 tested. Briefly, paths were removed between independent variables and
 eggs, then between independent variables and life history traits, and then
 between eggs and life history traits. We also ran the same model using λ’,
 a composite index that acts as a surrogate of population growth in place
 of survival, female mass, and development time. Unlike other studies that
 have considered only population growth we chose to focus on the individual
 life history variables for two reasons. First, the results were redundant
 in that we found similar results for λ’ to using each life history
 variable and survival, separately (not shown). And second, as traits could
 be affected independently by the environmental factors, using a surrogate
 that incorporated all three might mask important patterns. The final model
 for each species thus will represent all important paths included in the
 model to explain variation in oviposition and larval survival and could
 then be compared to our three hypothesized models to assess the importance
 of ecological filters in shaping patterns of these important
 insects.
description: One replicate was dropped for the <em>Culex
 quniquefasciauts</em> ovipostioin response to volume as no eggs were
 laid. No other data issues exist.