Many insect species produce nutritive ejaculates, which represent the male’s contribution to female reproductive fitness. Studies on the quantification of male contribution are assessed via indirect observations and direct assessment through the post-copulation effect on female reproductive performance, such as longevity and fecundity. Few studies directly address the nutritive content transferred by males via spermatophores. In species with multiple copulations, males produce ejaculates whose proportion of allocated substances varies according to the adult diet. We hypothesized that the spermatophores of C. montrouzieri show significant variations in nutrient composition when dietary modifications occur and whether this affects female fecundity and fertility. To test this hypothesis, we analyzed the macronutrient proteins, lipids, sugars, and glycogen using colorimetric methods. We tested for quantitative changes in these macronutrients in adult males well-fed with Planococcus citri mealybugs, under limited amounts of P. citri (food scarcity), and with honey. The macronutrient profile of a spermatophore from a well-fed C. montrouzieri is composed of approximately 49.62 µg/ml lipids, 5.18 µg/ml glycogen, 3.25 µg/ml sugar, and 0.27 µg/ml proteins. When compared to the spermatophores produced by males subjected to food scarcity or honey, no significant difference was found in the macronutrients. Fecundity was not affected, but there was a significant reduction in fertility in females that mated with males fed with honey.

A colony of C. montrouzieri was established at the Laboratório de Entomologia Aplicada (LEA) at the Universidade Federal de Pernambuco (UFPE). The individuals were maintained under constant conditions of 25 ± 1°C, 40 ± 10% relative humidity, and a 12-hour photoperiod. Larvae and adults C. montrouzieri were fed on adult females of Planococcus citri (Family: Pseudococcidae). Colonies of P. citri were kept in the laboratory and fed commercial "jacarezinho" pumpkins (Cucurbita moschata). Last-instar larvae of C. montrouzieri were separated and placed individually in Petri dishes (8 cm × 1.5 cm). Upon adult emergence (<24 hours), they were sexed and separated by gender for use in experiments.

Adult Diets

Females used in the experiments were maintained on a standard diet of adult P. citri females, ad libitum. Males were subjected to different dietary treatments: 1) Well-fed: males were fed adult females of P. citri, ad libitum. 2) Food scarcity: males were fed two adult females of P. citri every two days, based on preliminary tests to determine the minimum prey amount necessary to ensure male survival until the end of the experiments. 3) Honey: males were fed only commercial honey (Apinário Zumbi dos Palmares®; 70 kcal and 18 g of carbohydrates per 20 g portion, with negligible amounts of proteins, total fats, saturated fats, trans fats, dietary fiber, and sodium) at a 30% concentration, soaked in hydrophilic cotton.

Obtaining spermatophores

Males were fed their respective diets for 7 days prior to the start of the experiments. After the feeding period, males from different treatments were paired with females in cylindrical acrylic containers (1.9 cm × 3.4 cm) for copulation.  A copulation was considered complete when the male inserted his aedeagus into the female and remained in the mating position for at least 15 minutes before withdrawing voluntarily. If copulation did not occur during the observation period (6 consecutive hours), the adults were separated and re-paired after 24 hours. This procedure was repeated three times. Females that did not copulate were discarded.

To determine whether diets could affect the volume of spermatophores produced by males, a sample of spermatophores from well-fed (0,85 ± 0,18 mm³), food scarcity (1,12 ± 0,19 mm³)  and honey (1,11 ± 0,25 mm³) were measured (according to De Lima et al., 2022) and compared. No significant differences in spermatophore volume were found among treatments (Kruskal-Wallis test H₍₂₎ =0,8492; P= 0,6745).

Biochemical Analysis of Spermatophores

After complete copulations, females were dissected. For the quantitative biochemical analysis of spermatophores, total proteins were analyzed using the Bradford protocol (1976), and lipids, glycogen, and total sugars were analyzed using the Van Handel method (1985). Protocols were adjusted for accurate readings, given that C. montrouzieri spermatophores have a volume of 3.4 mm³ (De Lima et al., 2022). Preliminary experiments determined the minimum number of spermatophores required for accurate readings, based on the resolution of the available equipment to quantify the compounds. A pool of five spermatophores was used for each analysis. Each pool represented a repetition, with 10 repetitions per treatment, totaling 50 spermatophores per treatment.

Total Proteins

Protein quantification followed the Bradford protocol (1976). The calibration curve was standardized with bovine serum albumin (BSA). Spermatophores were transferred to a microtube containing 200 µl of sodium sulfate buffer (NaH₂PO₄H₂O; 0.1 M; pH 7.4) and manually macerated. Subsequently, 1000 µl of reagent was added, and samples were allowed to stand for 2 minutes at room temperature. Samples were analyzed using a Kasuaki® spectrophotometer, calibrated at 595 nm absorbance, and protein content was calculated based on a previously established standard curve.

Lipids, Sugars, and Glycogen

Quantification of lipids, glycogen, and sugars followed the Van Handel protocol (1985). The calibration curve for lipid analysis was standardized with commercial soybean oil, and the calibration curve for glycogen and sugar analysis was standardized with anhydrous glucose in deionized water. For macronutrient extraction, spermatophores were transferred to a microtube and manually macerated in 200 µl of sodium sulfate buffer. Next, 800 µl of chloroform/methanol solution was added and centrifuged (3000 rpm, 3 min). The supernatant was transferred to another clean microtube, and the pellet was used for glycogen analysis. To the supernatant, 600 µl of distilled water was added and centrifuged again (3000 rpm, 3 min). The upper fraction was used for sugar analysis and the lower fraction for lipid analysis.

Lipid Analysis: The lipid fraction was heated at 90°C until complete evaporation of the solvent, followed by the addition of 200 µl of sulfuric acid and further heating at 90°C for 10 minutes. After heating, 250 µl of vanillin reagent was added, gently mixed, and allowed to stand for 30 minutes. The reading was performed using a spectrophotometer at 625 nm absorbance, and lipid content was calculated based on a previously established standard curve.

Sugar Analysis: The sugar fraction was heated at 90°C until 200 µl of solvent remained, followed by the addition of 2500 µl of anthrone reagent, mixed slowly, and reheated for 17 minutes. After cooling, sugar analysis was performed using a spectrophotometer at 625 nm absorbance, and sugar content was calculated based on a previously established standard curve.

Glycogen Analysis: To the glycogen fraction, 2500 µl of anthrone reagent was added and lightly mixed, then heated at 90°C for 17 minutes. After cooling, samples were analyzed using a spectrophotometer at 625 nm absorbance, and glycogen content was calculated based on a previously established standard curve.

Fecundity and Fertility

To determine if the male diet affects female fecundity and fertility, males from each treatment (n= 30) were paired in cylindrical acrylic containers (1.9 cm × 3.4 cm) with females for copulation. After copulation, males were discarded, and females were individually placed in acrylic Petri dishes (60 × 15 mm) for oviposition. Egg counting began 24 hours after copulation. Eggs were counted daily for 10 days and transferred to new Petri dishes for fertility assessment. Egg masses were monitored for up to five days after laying, recording larval emergence. Eggs that did not hatch during this period were considered infertile.