DOI: 10.5061/dryad.vhhmgqp4c
title: Macronutrient composition of spermatophores of Cryptolaemus montrouzieri varies depending on male feeding

description: 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.
description: A colony of <em>C. montrouzieri</em> 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 <em>C. montrouzieri</em> were
 fed on adult females of <em>Planococcus citri</em> (Family:
 Pseudococcidae). Colonies of <em>P. citri</em> were kept in
 the laboratory and fed commercial "jacarezinho" pumpkins
 <em>(Cucurbita moschata</em>). Last-instar larvae of
 <em>C. montrouzieri</em> were separated and placed
 individually in Petri dishes (8 cm × 1.5 cm). Upon adult emergence
 (&lt;24 hours), they were sexed and separated by gender for use in
 experiments. <strong>Adult
 Diets</strong> Females used in the experiments
 were maintained on a standard diet of adult <em>P. citri</em>
 females, <em>ad libitum</em>. Males were subjected to
 different dietary treatments: 1) Well-fed: males were fed adult females of
 <em>P. citri</em>, <em>ad libitum</em>. 2) Food
 scarcity: males were fed two adult females of <em>P.
 citri</em> 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. <strong>Obtaining
 spermatophores</strong> 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 mm<sup>3</sup>),
 food scarcity (1,12 ± 0,19 mm<sup>3</sup>)  and honey (1,11 ±
 0,25 mm<sup>3</sup>) 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<sub>(2)</sub> =0,8492; P= 0,6745).
 <strong>Biochemical Analysis of
 Spermatophores</strong> 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 <em>C. montrouzieri</em>
 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<em> </em>of five
 spermatophores was used for each analysis. Each pool represented a
 repetition, with 10 repetitions per treatment, totaling 50 spermatophores
 per treatment. <strong>Total
 Proteins</strong> 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. <strong>Lipids,
 Sugars, and Glycogen</strong> 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. <strong>Lipid Analysis:</strong>
 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. <strong>Sugar Analysis:</strong> 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.
 <strong>Glycogen Analysis:</strong> 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.
 <strong>Fecundity and Fertility</strong>
 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.
description: # Macronutrient composition of spermatophores of *Cryptolaemus
 montrouzieri* varies depending on male feeding
 [https://doi.org/10.5061/dryad.vhhmgqp4c](https://doi.org/10.5061/dryad.vhhmgqp4c) ## Description of the data and file structure **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. ### Files and variables #### File: DATASET\_SENA\_ET\_AL.2.xlsx and DATASET\_SENA\_ET\_AL.2.csv **Description:** Colorimetric analysis obtained from 10 pools, each composed of five spermatophores, for the quantification of glycogen, sugar, proteins, and lipids. The readings were taken using a spectrophotometer and are presented in µg/mL. **MACRONUTRIENT ANALYSIS**: Includes the biochemical analyses of macronutrients. **FECUNDITY AND FERTILITY**: Contains data on egg-laying and larval hatching over a 30-day period (n), with values expressed as percentages. **CALIBRATION CURVE**: Includes the data used to standardize macronutrient curves, with one column for **OD** (which means the macronutrient value used for standardization) and another column for **ABS** (which means the absorbance number of the spectrophotometer). ## Code/software Software: *Microsoft 365* Version: 18.2503.1198.0