**4. Studies of sexual dimorphism in antennae of Mexican species of P***hyllophaga*

Sexual chemical communication in some Melolonthidae involves the production and release of specific chemicals by the emitter and the detection and olfactory processing of these signals leading to appropriate behavioral responses in the receiver [25]. Chemicals released from melolonthid females are captured in the sensilla located on both sides of male antennal lamellae [21,18,26]. The genus *Phyllophaga* is formed by more than 800 species, but only in *Phyllophaga anxia* LeConte [27] and in the antennae of Mexican species *Phyllophaga obsoleta* Blanchard [23] and *Phyllophaga ravida* Blanchard [24] have the different types of sensilla been studied. In the following sections data from *P. obsoleta* and *P. ravida* are remembered and compared with the data of another species of *Phyllophaga* distributed in Mexico.

#### **4.1. Methodology for the study of antennae of Mexican melolonthids**

#### *4.1.1. Measurement of body, antennae and lamellae dimensions*

After taxonomical identification using the keys proposed by Morón [18], females and males of *P. obsoleta*, *P. ravida* and *Phyllophaga opaca* Moser were randomly chosen for length measurement with the Image Tool 3.0 software program [28]. Length was measured in each specimen from the clypeus to the pygidium. The head of each previously measured specimen was separated from the body and preserved in 70% ethanol. The antennae of females and males of each species were separated and measurements of total length, width, and area of each lamella, were obtained with the Image Tool 3.0 software program. Afterwards, the three lamellae forming each antennal club were separated, labeled, and grouped according to sex and lamellar side (internal or external surfaces). The lamella located farther away from the head was denominated distal lamella (DL), while the nearest was called proximal lamella (PL) and the one between these two, middle lamella (ML).

#### *4.1.2. Specimen preparation for light microscopy studies*

Antennae from females and males of each species were kept in 10% KOH at 80ºC for 60 min, rinsed in distilled water, and placed in 70% ethanol in order to separate the lamellae, which were then dehydrated in 80%, 90%, and absolute ethanol. Lamellae were placed in xylene during 10 min for clearing, mounted in Canadian balsam and observed under a light microscope (Iroscope, Mod. MG-11J). Images from these slides and from non-cleared lamellae were obtained with a photo-microscope III (Carl Zeiss) and a Tessovar microscope (Carl Zeiss), both including a PaxCam 3 digital camera.

Sexual Dimorphism in Antennae of Mexican Species of *Phyllophaga* 

**Figure 2.** Habitus of species of *Phyllophaga*. *Phyllophaga obsoleta*, (A) male; (B) female. *Phyllophaga ravida*, (C) male; (D) female. *Phyllophaga opaca,* (E) male; (F) female. Scale lines 5 mm. Photos M. A. Morón.

(Coleoptera: Scarabaeoidea: Melolonthidae) 23

#### *4.1.3. Specimen preparation for scanning electron microscopy studies*

Lamellae from additional specimens were prepared following the methods proposed by [29] and were examined at 25kV under a scanning electron microscope (JEOL Mod. JSM-5600LV).

#### *4.1.4. Statistics*

Data on body, antennae and lamellae dimension for *P. opaca*, *P. ravida* and *P. obsoleta*, and adult males and females were analyzed with Student's t or Mann-Whitney Rank Sum tests (SigmaStat 3.1; 3.5). Unless otherwise stated, all values reported are mean standard error.

#### **5. Sexual dimorphism in antennae of** *Phyllophaga obsoleta*

This is one of the most noxious *Phyllophaga* species in México [30]. *Phyllophaga obsoleta* female's body length is larger than males, but the lamellar club is significantly longer and wider in males than females (Figures 2 A-B, Table 1)**.** In males, PL and ML were longer and had a larger area than in females (Table 1). Also, DL in males were longer, had a larger area and greater width than in females (Table 1).

Classification and terminology of sensilla types used here are based principally on [31] and in part on [21,32,27]. Six types of sensilla in both surfaces of antennal lamellae of both sexes of *P. obsoleta* were identified: sensilla placodea (PLAS), sensilla auricilica (AUS), sensilla basiconica (BAS), sensilla coeloconica (COS), sensilla trichodea (TRS) and sensilla chaetica (CHS). In Figure 3, these different sensilla types are showed. PLAS are thinwalled plates or are low dome shaped and BAS are cone-shaped. AUS are characterized by a "rabbit-ear" shape. COS are rarely found as aggregations of long rods located inside cuticle cavities. COS are observed only on internal surfaces of all lamellae from both sexes. TRS have a long hair-like structure that occurs along the peripheral edges while CHS present a short-bristle- or spine-like structure; they occur along the peripheral edges and some are in the lamellar center. According with above cited authors, TRS and CHS types are most likely mechano-receptors, but PLAS, AUS, BAS, and COS are considered chemo-receptor types.

5600LV).

error.

*4.1.4. Statistics* 

*4.1.2. Specimen preparation for light microscopy studies* 

(Carl Zeiss), both including a PaxCam 3 digital camera.

*4.1.3. Specimen preparation for scanning electron microscopy studies* 

**5. Sexual dimorphism in antennae of** *Phyllophaga obsoleta*

and greater width than in females (Table 1).

chemo-receptor types.

Antennae from females and males of each species were kept in 10% KOH at 80ºC for 60 min, rinsed in distilled water, and placed in 70% ethanol in order to separate the lamellae, which were then dehydrated in 80%, 90%, and absolute ethanol. Lamellae were placed in xylene during 10 min for clearing, mounted in Canadian balsam and observed under a light microscope (Iroscope, Mod. MG-11J). Images from these slides and from non-cleared lamellae were obtained with a photo-microscope III (Carl Zeiss) and a Tessovar microscope

Lamellae from additional specimens were prepared following the methods proposed by [29] and were examined at 25kV under a scanning electron microscope (JEOL Mod. JSM-

Data on body, antennae and lamellae dimension for *P. opaca*, *P. ravida* and *P. obsoleta*, and adult males and females were analyzed with Student's t or Mann-Whitney Rank Sum tests (SigmaStat 3.1; 3.5). Unless otherwise stated, all values reported are mean standard

This is one of the most noxious *Phyllophaga* species in México [30]. *Phyllophaga obsoleta* female's body length is larger than males, but the lamellar club is significantly longer and wider in males than females (Figures 2 A-B, Table 1)**.** In males, PL and ML were longer and had a larger area than in females (Table 1). Also, DL in males were longer, had a larger area

Classification and terminology of sensilla types used here are based principally on [31] and in part on [21,32,27]. Six types of sensilla in both surfaces of antennal lamellae of both sexes of *P. obsoleta* were identified: sensilla placodea (PLAS), sensilla auricilica (AUS), sensilla basiconica (BAS), sensilla coeloconica (COS), sensilla trichodea (TRS) and sensilla chaetica (CHS). In Figure 3, these different sensilla types are showed. PLAS are thinwalled plates or are low dome shaped and BAS are cone-shaped. AUS are characterized by a "rabbit-ear" shape. COS are rarely found as aggregations of long rods located inside cuticle cavities. COS are observed only on internal surfaces of all lamellae from both sexes. TRS have a long hair-like structure that occurs along the peripheral edges while CHS present a short-bristle- or spine-like structure; they occur along the peripheral edges and some are in the lamellar center. According with above cited authors, TRS and CHS types are most likely mechano-receptors, but PLAS, AUS, BAS, and COS are considered

**Figure 2.** Habitus of species of *Phyllophaga*. *Phyllophaga obsoleta*, (A) male; (B) female. *Phyllophaga ravida*, (C) male; (D) female. *Phyllophaga opaca,* (E) male; (F) female. Scale lines 5 mm. Photos M. A. Morón.


Sexual Dimorphism in Antennae of Mexican Species of *Phyllophaga* 

**6. Sexual dimorphism in antennae of** *Phyllophaga ravida*

of AUS, five of BAS, and four of COS.

TRS= sensilla trichodea; CHS= sensilla chaetica.

of males or females of *Phyllophaga ravida*.

*Phyllophaga ravida* is included in the "*dentex*" complex of the "*ravida*" species group, subgenus *Phyllophaga* (*sensu stricto*), and is one of the main white grub species of agricultural and economic importance in Mexico [30]. Several sex-related differences in antennae are observed. Although the body of adult *P. ravida* females is larger than that of males, the entire male antenna is considerably longer (Figures 2 C-D, Table 1). In males, DL is longer, wider, and cover a larger area than in females. Furthermore, the male ML is longer, with a larger area and greater width. Finally, male PL is longer, with a larger area and greater width (Table 1). Several types of sensilla were observed on *P. ravida* antennae using light- and scanning electron-microscopy: PLAS, AUS, BAS, COS, TRS and CHS (Table 2). TRS have a long spine- or hair-like structure and CHS present a short-spine, being shorter than TRS (Table 2). For *P. ravida*, sixteen chemo-sensilla types were found: three types of PLAS, four

Morphological characteristics of each chemo-sensilla type are described in the Table 2.

**PLAS II** spherical or elliptical and thin-walled plates

**AUS III** "human-ear" shaped structures

**BAS I** large peg- or cone-shaped **BAS II** short-spine shaped **BAS III** short peg- or cone shaped **BAS IV** serrated-cone shaped **BAS V** long-rod shaped

**COS III** long, rod-shaped aggregations (BAS V)

**TRS** long hair-like structure **CHS** short-bristle- or spine-like structure PLAS= sensilla placodea; AUS= sensilla auricilica; BAS= sensilla basiconica; COS= sensilla coeloconica;

along the peripheral edges of each lamella and some are in the lamellar center.

**AUS IV** small "rabbit-ear with neck" or "raisin with neck"

**PLAS III** small and elliptical thin-walled plates or low dome-shaped plates **AUS I** elliptical and thin walled-plates with "rabbit-ear" shape **AUS II** small "rabbit-ear" shaped structures, elliptical and

**COS I** aggregations of 2 to 16 long peg- or cone-shaped structures (BAS I) **COS II** aggregations of 2 to 3 serrated cone-shaped structures (BAS IV)

**COS IV** aggregations of a long cone (BAS I) and a spherical plate (PLAS I)

PLAS, AUS, BAS and COS are present on internal surface of lamellae while TRS and CHS are structures that occurs

**Table 2.** Morphological description of each of the types of antennal sensilla observed in the lamellae

low dome-shaped

shaped structures

**Sensilla types Morphological characteristics PLAS I** large spherical plates

(Coleoptera: Scarabaeoidea: Melolonthidae) 25

Values are mean ± standard error of the mean; *Student t*-test; *0=* not significant; (1)= Mann-Whitney Rank Sum test POBS: Phyllophaga obsoleta, n= 12; PRAV: Phyllophaga ravida, n= 14; POP: Phyllophaga opaca, n= 13; F= females; M= males; \* n=20; \*\*= n=6; PL = proximal lamella; ML = middle lamella; DL = distal lamella

**Table 1.** Comparison of the body and antennae dimensions, length, width and area of the lamellae of females and males of *Phyllophaga obsoleta, Phyllophaga ravida and Phyllophaga opaca.*

**Figure 3.** Scanning electron-micrograph of the internal surface (IN) of male *Phyllophaga obsoleta* middle lamella (ML). The IN is almost entirely covered by four sensillar types. In the periphery of the lamella, TRS and CHS are observed. B) TRS (long hair or setae shape). C) CHS (spine shape). D) PLAS (thinwalled plate or low dome), BAS (cone- or rod-shape) and AUS ("rabbit-ear-like") on the IN of the ML. E) Another view of PLAS, BAS and AUS. F) PLAS, BAS, AUS and COS (long rod aggregations located inside cuticle cavities). G) Detail of PLAS, BAS and COS. Micrographies by J. Valdez.

### **6. Sexual dimorphism in antennae of** *Phyllophaga ravida*

24 Sexual Dimorphism

**MEASURE (***mm***)**  

**Antennal** 

*POBS PRAV POP* 

**Body** 18.18 ±0.3 17.19 ±0.2 <0.05 22.13 ±0.3 21.17 ±0.2 <0.05 22.12 ± 0.2 21.16 ± 0.5 *0*  **Entire antennae** 3.30 ± 0.08 3.90 ± 0.04 <0.001 3.37 ±0.02 4.07 ±0.05 <0.001 2.88 ± 0.03 3.08 ± 0.05 <0.001

**flagellum** 2.15 ± 0.08 2.20 ± 0.04 *<sup>0</sup>*0.99 ±0.01 1.06 ±0.01 <0.05 1.80 ± 0.03 1.98 ± 0.04 <0.001 **Antennal club** 1.15 ± 0.02 1.70 ± 0.02 <0.001 1.15 ±0.01 1.71 ±0.02 <0.001 1.09 ± 0.01 1.10 ± 0.01 *0*  **PL length** 1.07 ± 0.02 1.66 ± 0.04 <0.001 1.19 ± 0.01 1.71 ± 0.04 <0.001 0.92 ± 0.02 0.95 ± 0.02 <0.05 **PL area (mm2)** 0.28 ± 0.01 0.51± 0.02 <0.001 0.38 ± 0.01 0.63 ± 0.02 <0.001 0.25 ± 0.01 0.26 ± 0.01 *0*  **PL width** 0.35 ± 0.01 0.38 ± 0.01 *0* 0.41 ± 0.01 0.46 ± 0.01 <0.05 0.35 ± 0.01 0.36 ± 0.01 *0*  **ML length** 1.10 ± 0.02 1.60 ± 0.03 <0.001 1.13 ± 0.01 1.67 ± 0.03 <0.001 (*<sup>1</sup>*) 0.81 ± 0.01 0.90 ± 0.02 <0.001 **ML area (mm2)** 0.29 ± 0.03 0.48 ± 0.04 <0.001 0.36 ± 0.01 0.61 ± 0.02 <0.001 0.19 ± 0.01 0.22 ± 0.01 <0.05 **ML width** 0.36 ± 0.01 0.37 ± 0.01 *0* 0.41 ± 0.01 0.47 ± 0.01 <0.05 0.30 ± 0.01 0.32 ± 0.01 <0.05 **DL length** 1.04 ± 0.03 1.58 ± 0.04 <0.001 1.04 ± 0.01 1.60 ± 0.03 <0.001 0.69 ± 0.02 0.82 ± 0.01 <0.001 **DL area (mm2)** 0.23 ± 0.01 0.44 ± 0.01 <0.001 0.31 ± 0.01 0.54 ± 0.01 <0.001 0.13 ± 0.02 0.17 ± 0.01 <0.001 **DL width** 0.31 ± 0.01 0.37 ± 0.01 <0.001 0.38 ± 0.01 0.42 ± 0.01 <0.001 0.24 ± 0.01 0.27 ± 0.01 <0.05

Values are mean ± standard error of the mean; *Student t*-test; *0=* not significant; (1)= Mann-Whitney Rank Sum test POBS: Phyllophaga obsoleta, n= 12; PRAV: Phyllophaga ravida, n= 14; POP: Phyllophaga opaca, n= 13; F= females; M=

**Table 1.** Comparison of the body and antennae dimensions, length, width and area of the lamellae of

**Figure 3.** Scanning electron-micrograph of the internal surface (IN) of male *Phyllophaga obsoleta* middle lamella (ML). The IN is almost entirely covered by four sensillar types. In the periphery of the lamella, TRS and CHS are observed. B) TRS (long hair or setae shape). C) CHS (spine shape). D) PLAS (thinwalled plate or low dome), BAS (cone- or rod-shape) and AUS ("rabbit-ear-like") on the IN of the ML. E) Another view of PLAS, BAS and AUS. F) PLAS, BAS, AUS and COS (long rod aggregations located

inside cuticle cavities). G) Detail of PLAS, BAS and COS. Micrographies by J. Valdez.

males; \* n=20; \*\*= n=6; PL = proximal lamella; ML = middle lamella; DL = distal lamella

females and males of *Phyllophaga obsoleta, Phyllophaga ravida and Phyllophaga opaca.*

**F M** *p* **F\* M\*** *p* **F\*\* M\*\*** *p* 

*Phyllophaga ravida* is included in the "*dentex*" complex of the "*ravida*" species group, subgenus *Phyllophaga* (*sensu stricto*), and is one of the main white grub species of agricultural and economic importance in Mexico [30]. Several sex-related differences in antennae are observed. Although the body of adult *P. ravida* females is larger than that of males, the entire male antenna is considerably longer (Figures 2 C-D, Table 1). In males, DL is longer, wider, and cover a larger area than in females. Furthermore, the male ML is longer, with a larger area and greater width. Finally, male PL is longer, with a larger area and greater width (Table 1). Several types of sensilla were observed on *P. ravida* antennae using light- and scanning electron-microscopy: PLAS, AUS, BAS, COS, TRS and CHS (Table 2). TRS have a long spine- or hair-like structure and CHS present a short-spine, being shorter than TRS (Table 2). For *P. ravida*, sixteen chemo-sensilla types were found: three types of PLAS, four of AUS, five of BAS, and four of COS.


Morphological characteristics of each chemo-sensilla type are described in the Table 2.

PLAS= sensilla placodea; AUS= sensilla auricilica; BAS= sensilla basiconica; COS= sensilla coeloconica; TRS= sensilla trichodea; CHS= sensilla chaetica.

PLAS, AUS, BAS and COS are present on internal surface of lamellae while TRS and CHS are structures that occurs along the peripheral edges of each lamella and some are in the lamellar center.

**Table 2.** Morphological description of each of the types of antennal sensilla observed in the lamellae of males or females of *Phyllophaga ravida*.

For both sexes, PLAS type I were observed randomly distributed on internal surface (IN) of all lamellae mainly in the center, except on peripheral edges of lamellae. PLAS type II were observed randomly distributed on IN and at the peripheral edges of all lamellae in both male and females. PLAS type III were located on IN of all lamellae, principally on pit, basal, and peripheral edges in both sexes. AUS type I were observed randomly distributed on IN and peripheral edges of all lamellae for both sexes. AUS type II was observed only in male lamellae, distributed on both sides (except on the external surface of the DL). AUS type III was located only on male lamellae, randomly distributed on IN and on peripheral edges (except on the IN of DL). AUS type IV were restricted to the center of PL in males. BAS type I were observed randomly distributed mostly on IN of all club lamellae of both sexes (except on peripheral edges and on the IN of ML). BAS type II are present for both sexes, they are situated on IN and peripheral edges of all lamellae. For both sexes, BAS type III were observed randomly distributed principally on IN of all club lamellae, except on peripheral edges. BAS type IV were found only in females at the center of the IN of ML and PL. BAS type V were found only in males, mainly at the center of the IN of PL. For both sexes, COS type I are restricted principally to the center, appearing only at the IN of all club lamellae. COS type II are located in both sexes, on the floor of cuticle cavities. COS type II are restricted only to females, mostly at the center and only on the IN of all club lamellae, except on DL. COS type III are located inside cavities in the antennal cuticle. COS III are restricted principally to the center, appearing only on the IN of all club lamellae in both sexes, except on male PL. COS type IV are found only in males and are restricted to the center and IN of PL. They are located inside depressions or cavities in the antennal cuticle.

Sexual Dimorphism in Antennae of Mexican Species of *Phyllophaga* 

**Figure 4.** Sensilla on the antennae of female *Phyllophaga opaca*. A) Internal surface (IN) of distal lamella (DL). In the periphery of the lamella, trichodea (TRS) and chaetica sensillum (CHS) are observed. B) Detail of placodea sensilla (PLAS) type III on external (EX) of proximal lamella (PL). C) Coeloconica sensilla (COS) type I, auricilica sensilla (AUS) type I, basiconica sensilla (BAS) type IV and PLAS I on IN of DL. D) Detail of COS I, PLAS I y PLAS II on IN of medium lamella (ML). E) COS I and BAS I on IN of

PL. F) Detail of COS I, AUS IV and BAS III on IN of PL. Micrographies by T. Laez.

(Coleoptera: Scarabaeoidea: Melolonthidae) 27
