INSECTS

Initially, S. acupunctatus larvae were obtained from tuberose crops in Mazatepec, (18.7334778°N, 99.3172222°W) and Yautepec (18.8289444°N, 99.09291667°W), Morelos, Mexico, from Dec 2015 to Dec 2016 and reared on tuberose bulbs in the laboratory. Upon emergence, adults were maintained in an environmental chamber at 25 ± 2 °C, 60% RH, 12:12 h (L:D) photoperiod. Individuals were separated by sex based on the criteria of Ramírez-Choza (1993) and females were kept individually in 30 mL plastic containers.

OVARIAN MATURATION

To determine the effect of diet on ovarian maturation, virgin S. acupunctatus were fed a meridic diet composed of agar, vitamins, sucrose, brewer's yeast, wheat bran, cholesterol, Wesson's salts, sorbic acid, ascorbic acid, methyl p-hydroxybenzoate, and distilled water (Valdés-Estrada et al. 2012). Another cohort was fed 5 mm wide tuberose bulb slices, previously hydrated in water for 20 min.

All females were dissected at 0, 5, 10, 15, 20, 30, 45, and 60 d of age (n = 12 for each age) under a stereoscopic microscope (Zeiss Discovery-V20, Thornwood, New York, USA). Ovaries were removed by pulling the ovipositor in a posterior direction until they were freed from the other tissues. Samples were stored at 4 °C in 5 mL Eppendorf tubes with water and thymol (May & Baker Ltd., Ongar, United Kingdom) solution of 0.5 mg per mL. Photomicrographs of dissected material were prepared using a Canon EOS 5D Mark II camera (Canon, Inc., Tokyo, Japan).

The proximal follicle area, length, and presence of mature follicles or relics were obtained from micrographs of dissected material. Only data from an ovariole of each weevil with the largest number of follicles were recorded. Length and area of the proximal follicle in each ovariole was measured using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). Follicle area was calculated using the formula of an ellipse (πab, where π = 3.1416, a = length/2, and b = width/2) (Perez-Mendoza et al 2004).

Ovarian development was classified based on the degree of follicular differentiation, maturity of the proximal follicle, and presence of follicular relics (or mature follicles) in the oviducts as reported by Grodowitz & Brewer (1987). Development was divided into 1 parous and 3 nulliparous stages. Nulliparous stages were classified as: nulliparous 1 (where the germarium and vitellarium are not differentiated and are without follicles); nulliparous 2 (well-defined ovarioles with multiple follicles, the proximal follicle transparent indicating that it had not completed maturation); and nulliparous 3 (proximal follicle more opaque [mature] and close to ovulation). In the parous stage the follicles pass through the lateral oviduct (ovulation) or have follicular relics at the base of the ovarioles.

STATISTICAL ANALYSIS

The mean number of follicles, as affected by diet and age (as well as mean length and area of the proximal follicle in each ovariole), was subjected to a 2-way ANOVA and differences determined by Tukey's test (P < 0.05). Females (on d of emergence) did not contain follicles, so this age was not included in analysis. Therefore, only length and area of the proximal follicle of individuals ≥⃒ 10 d of age were included in the analysis (75% females possessed follicles). All analyses were performed using SigmaPlot 12.5 (Systat Software, Inc., San Jose, California, USA).

OVARIAN MATURATION

We found that the female reproductive system of virgin S. acupunctatus was composed of a pair of ovaries with 2 tubular ovarioles each (Fig. 1). Only 0.01% of the dissected females possessed an ovary with 3 ovarioles; when this occurred the ovary was not fully developed and possessed no mature follicles. Follicle oocytes were surrounded by a layer of follicular cells that constituted the follicular epithelium (Fig. 2A). Follicles were observed to develop gradually into the vitellarium, where the least mature follicles were located distally whereas the mature ones occurred proximally, close to the common oviduct (Fig. 2B). Mature follicles were filled with yolk that contained a germinal vesicle or defined nucleus (Fig. 2C).

Ovarian development of virgin S. acupunctatus consisted of 4 stages: 3 nulliparous (without ovulation) and 1 parous (with ovulation) (Fig. 3). Ovulation occurred when the mature oocyte passed through the lateral oviduct (Fig. 3 PA). Follicle reabsorption and ovulation can be estimated by the presence of follicular relics at the base of the ovariole (Fig. 3 PB). Follicular relics were primarily light yellow, spotted, or ring shaped whereas very few were dark brown.

Females fed the meridic diet experienced a delay in ovariole maturation compared with those fed on tuberose bulbs (Fig. 4). We noted that all females fed the meridic diet at 0 and 5 d of age possessed undifferentiated ovaries without follicles (nulliparous 1), whereas 17% of those that fed on tuberose bulbs at 5 d of age presented well-defined ovarioles with follicles (nulliparous 2). At 10 d, 75% of females fed the meridic diet were in stage nulliparous 2 as well as 83% of females fed on tuberose; however, the rest of the females (17%) had mature follicles close to ovulation (nulliparous 3). At 15 d, 92% of the females fed on tuberose had already ovulated or had follicular relics (parous), but only 17% of the females fed on a meridic diet reached that stage (Fig. 4). At 20 d of age, all females fed on tuberose reached the parous stage, whereas 17% of those fed the meridic diet were in nulliparous 3 stage then later reached the parous stage at 30 d.

We also found that the number of follicles found in ovaries depended on the type of diet (F = 31.22; df = 1, 154; P < 0.001), the age of the individual (F = 76.71; df = 6, 154; P < 0.01), and the interaction between these 2 variables (F = 2.42; df = 6, 154; P < 0.05). Females fed tuberose bulbs gradually increased their number of follicles up through 20 d of age followed by a decrease; individuals fed the meridic diet showed similar development but with a delay of 10 d (Fig. 5). The production of follicles in females fed on tuberose was significantly greater than that of females fed on a meridic diet at 10, 20, and 45 d of age.

Length of the proximal follicle of individual weevils containing the most mature oocyte (Fig. 6A) was not affected by diet (F = 2.73; df = 1, 129; P > 0.05), but was affected by age (F = 22.75; df = 5, 129; P < 0.001), as well as the interaction between these 2 factors (F = 3.16; df = 5, 129; P = 0.010). The length of the proximal follicle increased progressively with respect to age until 30 and 20 d of age in females fed the meridic diet and tuberose bulb, respectively. At 30 d of age, the proximal follicle length of females fed the meridic diet was significantly greater compared with individuals that fed on tuberose (Fig. 6A); however, at 45 d of age this relationship was reversed.

The proximal follicular area was not affected by diet (F = 0.05; df = 1, 119; P > 0.05) but was affected by age (F = 19.21; df = 5, 129; P < 0.001), and interaction of diet and age (F = 4.24; df = 5, 129; P < 0.001). The proximal follicle area increased gradually with time until 30 and 20 d of age in individuals fed on the meridic diet and tuberose bulbs, respectively (Fig. 6B). Maximum increase was observed at 30 and 60 d of age in females fed the meridic diet, whereas those fed on tuberose was 45 d. Moreover, females that fed on tuberose bulbs had a significantly smaller proximal follicle area than those fed the meridic diet at 30 d, reversing this relationship at 45 d of age.

Fig. 1.

(A) Female reproductive system of Scyphophorus acupunctatus formed by 2 ovaries (ov), germarium (g), vitellarium (v), oocyte (o), lateral oviduct (lo), common oviduct (co), spermatheca (s), and genital chamber (gc); (B) Distal part of the germarium (g) with bulky appearance possibly with trophocytes, prefollicular cells, and oogonia, terminal filament (tf), trachea (tr), tracheoles (trl); and (C) Approach to the onset of vitellarium with oocytes in development, germinal vesicle (gv), interfollicular tissue (tif), trachea (tr), and tracheoles (trl).